Gamma-heregulin

ABSTRACT

A novel member of the heregulin superfamily has been identified which is designated “γ-HRG”. This molecule, secreted by human breast cancer MDA-MB-175 cells, leads to the formation of a constitutive active receptor complex and stimulates the growth of these cells in an autocrine manner. γ-HRG polypeptide and nucleic acid are disclosed, together with various uses therefor (e.g. use of γ-HRG nucleic acid for the recombinant production of γ-HRG). γ-HRG antagonists (e.g. neutralizing antibodies and antisense nucleic acid molecules) as well as uses therefor are also described.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/173,893, filed on Jul. 1, 2005, which is a continuation ofapplication Ser. No. 10/290,578, filed on Nov. 8, 2002 (now U.S. Pat.No. 6,916,624), which is a divisional of U.S. application Ser. No.09/514,573 filed Feb. 28, 2000 (now U.S. Pat. No. 6,500,941), which is acontinuation of U.S. application Ser. No. 08/891,845 filed Jul. 10, 1997(now U.S. Pat. No. 6,096,873), which is a non-provisional applicationfiled under 37 CFR 1.53(b)(1), claiming priority under 35 USC Section119(e) to provisional U.S. Application Ser. No. 60/021,640 filed on Jul.12, 1996 (now abandoned), the disclosures of which are hereinincorporated by reference in their entirety.

FIELD OF THE INVENTION

This application relates to the discovery of a novel heregulinpolypeptide called gamma-heregulin (γ-HRG) secreted by human breastcancer MDA-MB-175 cells, which has a unique N-terminal domain notpresent in hitherto-identified heregulins.

BACKGROUND OF THE INVENTION

Transduction of signals that regulate cell growth and differentiation isregulated in part by phosphorylation of various cellular proteins.Protein tyrosine kinases are enzymes that catalyze this process.Receptor protein tyrosine kinases are believed to direct cellular growthvia ligand-stimulated tyrosine phosphorylation of intracellularsubstrates. Growth factor receptor protein tyrosine kinases of the classI subfamily include the 170 kDa epidermal growth factor receptor (EGFR)encoded by the erbB1 gene. erbB1 has been causally implicated in humanmalignancy. In particular, increased expression of this gene has beenobserved in more aggressive carcinomas of the breast, bladder, lung andstomach.

The second member of the class I subfamily, p185^(neu), was originallyidentified as the product of the transforming gene from neuroblastomasof chemically treated rats. The neu gene (also called erbB2 and HER2)encodes a 185 kDa receptor protein tyrosine kinase. Amplification and/oroverexpression of the human HER2 gene correlates with a poor prognosisin breast and ovarian cancers (Slamon et al., Science 235:177-182(1987); and Slamon et al., Science 244:707-712 (1989)). Overexpressionof HER2 has been correlated with other carcinomas including carcinomasof the stomach, endometrium, salivary gland, lung, kidney, colon andbladder. Accordingly, Slamon et al. in U.S. Pat. No. 4,968,603 describeand claim various diagnostic assays for determining HER2 geneamplification or expression in tumor cells. Slamon et al. discoveredthat the presence of multiple gene copies of HER2 oncogene in tumorcells indicates that the disease is more likely to spread beyond theprimary tumor site, and that the disease may therefore require moreaggressive treatment than might otherwise be indicated by otherdiagnostic factors. Slamon et al. conclude that the HER2 geneamplification test, together with the determination of lymph nodestatus, provides greatly improved prognostic utility.

A further related gene, called erbB3 or HER3, has also been described.See U.S. Pat. No. 5,183,884; Kraus et al., Proc. Natl. Acad. Sci. USA86:9193-9197 (1989); EP Pat Appln No 444,961A1; and Kraus et al., Proc.Natl. Acad. Sci. USA 90:2900-2904 (1993). Kraus et al. (1989) discoveredthat markedly elevated levels of erbB3 mRNA were present in certainhuman mammary tumor cell lines indicating that erbB3, like erbB1 anderbB2, may play a role in human malignancies. Also, Kraus et al. (1993)showed that EGF-dependent activation of the ErbB3 catalytic domain of achimeric EGFR/ErbB3 receptor resulted in a proliferative response intransfected NIH-3T3 cells. Furthermore, these researchers demonstratedthat some human mammary tumor cell lines display a significant elevationof steadγ-state ErbB3 tyrosine phosphorylation further indicating thatthis receptor may play a role in human malignancies. The role of erbB3in cancer has been explored by others. It has been found to beoverexpressed in breast (Lemoine et al., Br. J. Cancer 66:1116-1121(1992)), gastrointestinal (Poller et al., J. Pathol. 168:275-280 (1992),Rajkumar et al., J. Pathol. 170:271-278 (1993), and Sanidas et al., Int.J. Cancer 54:935-940 (1993)), and pancreatic cancers (Lemoine et al., J.Pathol. 168:269-273 (1992), and Friess et al., Clinical Cancer Research1:1413-1420 (1995)).

The class I subfamily of growth factor receptor protein tyrosine kinaseshas been further extended to include the HER4/Erb4 receptor. See EP PatAppln No 599,274; Plowman et al., Proc. Natl. Acad. Sci. USA90:1746-1750 (1993); and Plowman et al., Nature 366:473-475 (1993).Plowman et al. found that increased HER4 expression closely correlatedwith certain carcinomas of epithelial origin, including breastadenocarcinomas. Diagnostic methods for detection of human neoplasticconditions (especially breast cancers) which evaluate HER4 expressionare described in EP Pat Appln No. 599,274.

The quest for the activator of the HER2 oncogene has lead to thediscovery of a family of heregulin polypeptides. These proteins appearto result from alternate splicing of a single gene which was mapped tothe short arm of human chromosome 8 by Orr-Urtreger et al., Proc. Natl.Acad. Sci. USA 90:1867-1871 (1993).

Holmes et al. isolated and cloned a family of polypeptide activators forthe HER2 receptor which they called heregulin-α(HRG-α), heregulin-β1(HRG-β1), heregulin-β2 (HRG-β2), heregulin-β2-like (HRG-β2-like), andheregulin-β3 (HRG-β3). See Holmes et al., Science 256:1205-1210 (1992);WO 92/20798; and U.S. Pat. No. 5,367,060. The 45 kDa polypeptide, HRG-α,was purified from the conditioned medium of the MDA-MB-231 human breastcancer cell line. These researchers demonstrated the ability of thepurified heregulin polypeptides to activate tyrosine phosphorylation ofthe HER2 receptor in MCF7 breast tumor cells. Furthermore, the mitogenicactivity of the heregulin polypeptides on SK-BR-3 cells (which expresshigh levels of the HER2 receptor) was illustrated. Like other growthfactors which belong to the EGF family, soluble HRG polypeptides appearto be derived from a membrane bound precursor (called pro-HRG) which isproteolytically processed to release the 45 kDa soluble form. Thesepro-HRGs lack a N-terminal signal peptide.

While heregulins are substantially identical in the first 213 amino acidresidues, they are classified into two major types, α and β, based ontwo variant EGF-like domains which differ in their C-terminal portions.Nevertheless, these EGF-like domains are identical in the spacing of sixcysteine residues contained therein. Based on an amino acid sequencecomparison, Holmes et al. found that between the first and sixthcysteines in the EGF-like domain, HRGs were 45% similar toheparin-binding EGF-like growth factor (HB-EGF), 35% identical toamphiregulin (AR), 32% identical to TGF-α, and 27% identical to EGF.

The 44 kDa neu differentiation factor (NDF), which is the rat equivalentof human HRG, was first described by Peles et al., Cell, 69:205-216(1992); and Wen et al., Cell, 69:559-572 (1992). Like the HRGpolypeptides, NDF has an immunoglobulin (Ig) homology domain followed byan EGF-like domain and lacks a N-terminal signal peptide. Subsequently,Wen et al., Mol. Cell. Biol., 14(3):1909-1919 (1994) carried out“exhaustive cloning” to extend the family of NDFs. This work revealedsix distinct fibroblastic pro-NDFs. Adopting the nomenclature of Holmeset al., the NDFs are classified as either α or β polypeptides based onthe sequences of the EGF-like domains. Isoforms 1 to 4 are characterizedon the basis of the variable just a membrane stretch (between theEGF-like domain and transmembrane domain). Also, isoforms a, b and c aredescribed which have variable length cytoplasmic domains. Theseresearchers conclude that different NDF isoforms are generated byalternative splicing and perform distinct tissue-specific functions. Seealso EP 505 148; WO 93/22424; and WO 94/28133 concerning NDF.

Falls et al., Cell, 72:801-815 (1993) describe another member of theheregulin family which they call acetylcholine receptor inducingactivity (ARIA) polypeptide. The chicken-derived ARIA polypeptidestimulates synthesis of muscle acetylcholine receptors. See also WO94/08007. ARIA is a β-type heregulin and lacks the entire spacer regionrich in glycosylation sites between the Ig-like domain and EGF-likedomain of HRGα, and HRGβ1-β3.

Marchionni et al., Nature, 362:312-318 (1993) identified severalbovine-derived proteins which they call glial growth factors (GGFs).These GGFs share the Ig-like domain and EGF-like domain with the otherheregulin proteins described above, but also have an amino-terminalkringle domain. GGFs generally do not have the complete glycosylatedspacer region between the Ig-like domain and EGF-like domain. Only oneof the GGFs, GGFII, possessed a N-terminal signal peptide. See also WO92/18627; WO 94/00140; WO 94/04560; WO 94/26298; and WO 95/32724 whichrefer to GGFs and uses thereof.

Ho et al. in J. Biol. Chem. 270(24):14523-14532 (1995) describe anothermember of the heregulin family called sensory and motor neuron-derivedfactor (SMDF). This protein has an EGF-like domain characteristic of allother heregulin polypeptides but a distinct N-terminal domain. The majorstructural difference between SMDF and the other heregulin polypeptidesis the lack in SMDF of the Ig-like domain and the “glyco” spacercharacteristic of all the other heregulin polypeptides. Another featureof SMDF is the presence of two stretches of hydrophobic amino acids nearthe N-terminus.

While the heregulin polypeptides were first identified based on theirability to activate the HER2 receptor (see Holmes et al., supra), it wasdiscovered that certain ovarian cells expressing neu and neu-transfectedfibroblasts did not bind or crosslink to NDF, nor did they respond toNDF to undergo tyrosine phosphorylation (Peles et al., EMBO J.12:961-971 (1993)). This indicated another cellular component wasnecessary for conferring full heregulin responsiveness. Carraway et al.subsequently demonstrated that ¹²⁵I-rHRGβ1₁₇₇₋₂₄₄ bound to NIH-3T3fibroblasts stably transfected with bovine erbB3 but not tonon-transfected parental cells. Accordingly, they conclude that ErbB3 isa receptor for HRG and mediates phosphorylation of intrinsic tyrosineresidues as well as phosphorylation of ErbB2 receptor in cells whichexpress both receptors. Carraway et al., J. Biol. Chem.269(19):14303-14306 (1994). Sliwkowski et al., J. Biol. Chem.269(20):14661-14665 (1994) found that cells transfected with HER3 aloneshow low affinities for heregulin, whereas cell transfected with bothHER2 and HER3 show higher affinities.

This observation correlates with the “receptor cross-talking” describedpreviously by Kokai et al., Cell 58:287-292 (1989); Stem et al., EMBO J.7:995-1001 (1988); and King et al., Oncogene 4:13-18 (1989). Theseresearchers found that binding of EGF to the EGFR resulted in activationof the EGFR kinase domain and cross-phosphorylation of p185^(HER2). Thisis believed to be a result of ligand-induced receptor heterodimerizationand the concomitant cross-phosphorylation of the receptors within theheterodimer (Wada et al., Cell 61:1339-1347 (1990)).

Plowman and his colleagues have similarly studiedp185^(HER4)/p185^(HER2) activation. They expressed p185^(HER2) alone,p185^(HER4) alone, or the two receptors together in human T lymphocytesand demonstrated that heregulin is capable of stimulating tyrosinephosphorylation of p185^(HER4), but could only stimulate p185^(HER2)phosphorylation in cells expressing both receptors. Plowman et al.,Nature 366:473-475 (1993). Thus, heregulin is the only known example ofa member of the EGF growth factor family that can interact with severalreceptors. Carraway and Cantley, Cell 78:5-8 (1994).

The biological role of heregulin has been investigated by severalgroups. For example, Falls et al., (discussed above) found that ARIAplays a role in myotube differentiation, namely affecting the synthesisand concentration of neurotransmitter receptors in the postsynapticmuscle cells of motor neurons. Corfas and Fischbach demonstrated thatARIA also increases the number of sodium channels in chick muscle.Corfas and Fischbach, J. Neuroscience, 13(5):2118-2125 (1993). It hasalso been shown that GGFII is mitogenic for subconfluent quiescent humanmyoblasts and that differentiation of clonal human myoblasts in thecontinuous presence of GGFII results in greater numbers of myotubesafter six days of differentiation (Sklar et al., J. Cell Biochem., Abst.W462, 18D, 540 (1994)). See also WO 94/26298 published Nov. 24, 1994.

Holmes et al., supra, found that HRG exerted a mitogenic effect onmammary cell lines (such as SK-BR-3 and MCF-7). The mitogenic activityof GGFs on Schwann cells has also been reported. See, e.g., Brockes etal., J. Biol. Chem. 255(18):8374-8377 (1980); Lemke and Brockes, J.Neurosci. 4:75-83 (1984); Brockes et al., Ann. Neurol. 20(3):317-322(1986); Brockes, J., Methods in Enzym., 147:217-225 (1987) andMarchionni et al., supra. Schwann cells constitute important glial cellswhich provide myelin sheathing around the axons of neurons, therebyforming individual nerve fibers. Thus, it is apparent that Schwann cellsplay an important role in the development, function and regeneration ofperipheral nerves. The implications of this from therapeutic standpointhave been addressed by Levi et al., J. Neuroscience 14(3):1309-1319(1994). Levi et al. discuss the potential for construction of a cellularprosthesis comprising human Schwann cells which could be transplantedinto areas of damaged spinal cord. Methods for culturing Schwann cellsex vivo have been described. See WO 94/00140 and Li et al., J.Neuroscience 16(6):2012-2019 (1996).

Pinkas-Kramarski et al. found that NDF seems to be expressed in neuronsand glial cells in embryonic and adult rat brain and primary cultures ofrat brain cells, and suggested that it may act as a survival andmaturation factor for astrocytes (Pinkas-Kramarski et al., PNAS, USA91:9387-9391 (1994)). Meyer and Birchmeier, PNAS, USA 91:1064-1068(1994) analyzed expression of heregulin during mouse embryogenesis andin the perinatal animal using in situ hybridization and RNase protectionexperiments. These authors conclude that, based on expression of thismolecule, heregulin plays a role in vivo as a mesenchymal and neuronalfactor. Also, their findings imply that heregulin functions in thedevelopment of epithelia. Similarly, Danilenko et al., Abstract 3101,FASEB 8(4-5):A535 (1994), found that the interaction of NDF and the HER2receptor is important in directing epidermal migration anddifferentiation during wound repair.

SUMMARY OF THE INVENTION

The invention relates to the discovery of the novel γ-HRG polypeptideand nucleic acid. This molecule, secreted by human breast cancerMDA-MB-175 cells, leads to the formation of a constitutive activereceptor complex and stimulates the growth of these cells in anautocrine manner. Accordingly, the invention provides isolated γ-HRGpolypeptide. This γ-HRG polypeptide is preferably substantiallyhomogeneous and may be selected from the group consisting of a nativesequence polypeptide (such as human γ-HRG of FIG. 1) and variant γ-HRG(e.g. chimeric γ-HRG). Additionally, the γ-HRG polypeptide may beselected from the group consisting of the polypeptide that is isolatedfrom a mammal (e.g. a human), the polypeptide that is made byrecombinant means, and the polypeptide that is made by synthetic means.Accordingly, the polypeptide may be unassociated with nativeglycosylation or may be completely unglycosylated. In preferredembodiments, the isolated γ-HRG possesses an effector function of humanγ-HRG of SEQ ID NO:2 and comprises an amino acid sequence selected fromthe group consisting of: (a) the amino acid sequence for mature humanγ-HRG in SEQ ID NO:2; (b) the naturally occurring amino acid sequencefor mature γ-HRG from an animal species other than the sequence of (a);(c) naturally occurring allelic variants or isoforms of (a) or (b); and(d) the amino acid sequence of (a), (b) or (c) which has only one or twoamino acid substitutions.

The invention further provides a composition (preferably one which issterile) comprising γ-HRG and a pharmaceutically acceptable carrier. Thecomposition may be used in a method for activating an ErbB receptorwhich comprises the step of contacting a cell which expresses an ErbBreceptor (which may be the same or different from the ErbB receptor tobe activated) with the γ-HRG polypeptide. This method may be an in vitroone, e.g. where the cell is in cell culture or an in vivo method wherethe cell is present in a mammal (e.g. a human patient who could benefitfrom ErbB receptor activation). In another embodiment, the inventionprovides an in vitro or in vivo method for enhancing proliferation,differentiation or survival of a cell (especially where the cellexpresses an ErbB receptor at its cell surface) comprising the step ofcontacting the cell with the γ-HRG polypeptide. The cell may, forexample, be a glial cell or muscle cell. Furthermore, the inventionprovides a method for detecting an ErbB receptor which comprises thestep of contacting a sample suspected of containing the ErbB receptorwith the γ-HRG polypeptide (e.g. labelled γ-HRG) and detecting ifbinding has occurred. In this manner, an assay for determining aprognosis in patients suffering from carcinoma (e.g. breast or ovariancarcinoma) is provided.

γ-HRG has a unique N-terminal domain (NTD) which is not present in otherheregulins. This NTD and fragments thereof (as well the nucleic acidencoding NTD or fragments thereof) is thought to be particularly usefulfor the production of γ-HRG-specific reagents, e.g. anti-NTD antibodiesfor detecting and purifying γ-HRG as well as nucleic acid probes.Accordingly, the invention provides an isolated polypeptide comprising aconsecutive sequence of at least thirty amino acids of theγ-HRG-terminal domain (NTD) of SEQ ID NO:4 or one which comprises theamino acid sequence for mature γ-HRG-terminal domain (NTD) in SEQ IDNO:4.

The NTD-specific antibodies may be used, among other things, in a methodfor detecting γ-HRG which comprises the step of contacting a samplesuspected of containing γ-HRG with the antibody (which is optionallylabelled) and detecting if binding has occurred. The antibody may alsobe used in a method for purifying γ-HRG which comprises the step ofpassing a mixture containing γ-HRG over a solid phase to which is boundthe antibody and recovering the fraction containing γ-HRG.

The nucleic acid encoding the NTD of γ-HRG may be used to determine thepresence of a nucleic acid molecule encoding γ-HRG in a test sample(e.g. from a mammal suspected of having, or being predisposed tocancer), comprising contacting the test sample with the isolated nucleicacid and determining whether the isolated nucleic acid hybridizes to anucleic acid molecule in the test sample. Nucleic acid encoding the NTDof γ-HRG may also be used in hybridization assays to identify andisolate nucleic acids sharing substantial sequence identity to γ-HRG. Infurther embodiments, this NTD-encoding nucleic acid can be used as aprimer in a polymerase chain reaction for amplifying a nucleic acidmolecule encoding γ-HRG in a test sample.

The invention also provides γ-HRG-specific antagonists for use inmethods where it is desirable to block γ-HRG production and/orbiological activity either in vitro or in vivo. One type of antagonistis a neutralizing antibody which binds specifically to the NTD of γ-HRG.Another type of antagonist is an antisense nucleic acid molecule, e.g.one which is complementary to the nucleic acid sequence encoding the NTDand which is able to reduce production of γ-HRG polypeptide byMDA-MB-175 cells.

In other aspects, the invention provides an isolated nucleic acidmolecule encoding γ-HRG (and isolated antisense nucleic acid molecules;see above). For example, the nucleic acid molecule may be selected fromthe group consisting of: (a) nucleic acid comprising the nucleotidesequence of the coding region of the mature γ-HRG gene in SEQ ID NO:1;(b) nucleic acid corresponding to the sequence of (a) within the scopeof degeneracy of the genetic code; and (c) nucleic acid which hybridizesto DNA complementary to DNA encoding the N-terminal domain (NTD) ofhuman γ-HRG of SEQ ID NO:2 under moderately stringent conditions andwhich encodes a polypeptide possessing an effector function of humanγ-HRG of SEQ ID NO:2. The isolated nucleic acid molecule optionallyfurther comprises a promoter operably linked thereto.

In other embodiments, the invention provides a vector comprising thenucleic acid molecule (e.g. an expression vector comprising the nucleicacid molecule operably linked to control sequences recognized by a hostcell transformed with the vector); a host cell comprising the nucleicacid molecule; and a method of using a nucleic acid molecule encodingγ-HRG to effect production of γ-HRG which comprises the step ofculturing the host cell and recovering γ-HRG from the cell culture. Theisolated nucleic acid may also be used for in vivo or ex vivo genetherapy.

As an alternative to production of the γ-HRG in a transformed host cell,the invention provides a method for producing γ-HRG comprising: (a)transforming a cell containing an endogenous γ-HRG gene with ahomologous DNA comprising an amplificable gene and a flanking sequenceof at least about 150 base pairs that is homologous with a DNA sequencewithin or in proximity to the endogenous γ-HRG gene, whereby thehomologous DNA integrates into the cell genome by recombination; (b)culturing the cell under conditions that select for amplification of theamplifiable gene, whereby the γ-HRG gene is also amplified; andthereafter (c) recovering γ-HRG from the cell.

The invention further provides a method for treating a mammal comprisingadministering a therapeutically effective amount of γ-HRG to the mammal.For example, the mammal may be suffering from a neurological or musculardisorder. Conversely, the invention provides a method for treating amammal comprising administering to a therapeutically effective amount ofa γ-HRG antagonist to the mammal. The mammal in this latter case is onewhich could benefit from a reduction in γ-HRG levels/biological activity(e.g. in cancer).

These and other aspects of the invention will be apparent to thoseskilled in the art upon consideration of the following detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C depict the cDNA (SEQ ID NO:1) and deduced amino acid sequence(SEQ ID NO:2) of γ-HRG. The hydrophobic region is underlined. TheEGF-like domain is shaded, cysteine residues in the EGF-like domain arecircled. N-linked glycosylation sites are marked above the nucleic acidsequence with a (•).

FIG. 2 is a schematic comparison of different HRG isoforms. Boxesrepresent major structural motifs of various HRG isoforms. Thestructural features of γ-HRG are compared with HRGβ, SMDF and GGF. TheEGF-like domain (EGF) is shown as a black box. The juxtamembrane(numbered), the transmembrane (TM) region and the cytoplasmic domain aredrawn as distinct boxes. The glycosylated spacer domain (Spacer domain)separates the EGF-like domain from the Ig-like domain (Ig domain) inγ-HRG, HRGβ and SMDF. The unique N-terminal sequence of γ-HRG isstriped. The N-terminal regions of HRGβ, SMDF and GGF are differentlyshaded. GGF possesses a kringle-like motif (kringle) and a signalpeptide sequence (SP) in the N-terminal region.

FIG. 3 is a displacement curve of ¹²⁵I-rHRGβ1₍₁₇₇₋₂₄₄₎ binding withunlabeled γ-HRG. ErbB3- or ErbB4-immunoadhesins were incubated with¹²⁵I-rHRGβ1₍₁₇₇₋₂₄₄₎ (0.23 nM) and various amounts of γ-HRG.

FIGS. 4A and 4B show the effect of 2C4, 4D5 and ErbB4 immunoadhesins onproliferation of MDA-MB-175 (FIG. 4A) and SK-BR-3 (FIG. 4B) cells.MDA-MB-175 and SK-BR-3 cells were seeded in 96 well plates and allowedto adhere for 2 hours. Experiment was carried out in medium containing1% serum. Anti-ErbB2 antibodies, ErbB4-immunoadhesin (H4-IgG) or mediumalone were added and the cells were incubated for 2 hours at 37° C.Subsequently rHRGβ1 (1 nM) or 100 nM rHRGβ1 for neutralizing the H4-IgGeffect or medium alone were added and the cells were incubated for 4days. Monolayers were washed and stained/fixed with 0.5% crystal violet.To determine cell proliferation the absorbance was measured at 540 nm.

FIGS. 5A-B are an alignment of the γ-HRG amino acid sequence (SEQ IDNO:2) with the partial amino acid sequence (SEQ ID NO:10) of the γ-HRGisoform (clone 20) identified in the Example.

FIGS. 6A-G show an alignment of the γ-HRG nucleic acid sequence (SEQ IDNO:1) with the partial nucleotide sequence (SEQ ID NO:11) of the γ-HRGisoform (clone 20) identified in the Example.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definitions

In general, the following words or phrases have the indicated definitionwhen used in the description, examples, and claims.

Unless indicated otherwise, the term “ErbB” when used herein refers toany one or more of the mammalian ErbB receptors (i.e. ErbB 1 orepidermal growth factor (EGF) receptor; ErbB2 or HER2 receptor; ErbB3 orHER3 receptor; ErbB4 or HER4 receptor; and any other member(s) of thisclass I tyrosine kinase family to be identified in the future) and“erbB” refers to the mammalian erbB genes encoding these receptors.

“γ-HRG” (or “gamma-heregulin”) is defined herein to be any polypeptidesequence that possesses at least one biological property (as definedbelow) of native sequence γ-HRG of SEQ ID NO:2. This definitionencompasses not only the polypeptide isolated from a native γ-HRG sourcesuch as human MDA-MB-175 cells or from another source, such as anotheranimal species, but also the polypeptide prepared by recombinant orsynthetic methods. It also includes variant forms including functionalderivatives, allelic variants, naturally occurring isoforms andanalogues thereof. Sometimes the γ-HRG is “native γ-HRG” which refers toendogenous γ-HRG polypeptide which has been isolated from a mammal. Theγ-HRG can also be “native sequence γ-HRG” insofar as it has the sameamino acid sequence as a native γ-HRG (e.g. human γ-HRG shown in FIG.1). However, “native sequence γ-HRG” encompasses the polypeptideproduced by recombinant or synthetic means. “Mature γ-HRG” is soluble orsecreted γ-HRG released from the cell (i.e. lacking amino-terminalsequence). γ-HRG “isoforms” are naturally occurring polypeptides whichcomprise at least part of the N-terminal domain (see below) of γ-HRG. Anexample of a γ-HRG isoform is described in the Example below.

Optionally, the γ-HRG is not associated with native glycosylation.“Native glycosylation” refers to the carbohydrate moieties which arecovalently attached to native γ-HRG when it is produced in the mammaliancell from which the native γ-HRG is derived. Accordingly, human γ-HRGproduced in a non-human cell could be described as not being associatedwith native glycosylation, for example. Sometimes, the γ-HRG is notassociated with any glycosylation whatsoever (e.g. as a result of beingproduced recombinantly in a prokaryote).

γ-HRG has a unique amino terminal domain which distinguishes thisprotein from previously described heregulin polypeptides. This isdesignated the “N-terminal domain” or “NTD” herein (i.e. from aboutresidue 1 to about residue 560 of FIG. 1 (SEQ ID NO:4 encoded by thenucleic acid sequence of SEQ ID NO:3)). “Mature” NTD is NTD releasedfrom the cell surface. However, the expression “NTD” includes functionalequivalents of the NTD depicted in FIG. 1.

The term “γ-HRG variant” encompasses γ-HRG polypeptides wherein one ormore amino acid residues are added at the—or C-terminus of, or within,the γ-HRG sequence of FIG. 1; one or more amino acid residues aredeleted, and optionally substituted by one or more amino acid residues;and derivatives of the above polypeptides, wherein an amino acid residuehas been covalently modified so that the resulting product has anon-naturally occurring amino acid. Ordinarily, a biologically activeγ-HRG variant will be “substantially homologous” to the amino acidsequence of FIG. 1 and therefore will generally have an amino acidsequence having at least about 70% amino acid sequence identity withhuman γ-HRG shown in FIG. 1, preferably at least about 75%, morepreferably at least about 80%, still more preferably at least about 85%,even more preferably at least about 90%, and most preferably at leastabout 95%.

One type of γ-HRG variant is a “γ-HRG fragment”, which is a portion of anaturally occurring full-length γ-HRG sequence having one or more aminoacid residues or carbohydrate units deleted. The deleted amino acidresidue(s) may occur anywhere in the polypeptide, including at eitherthe N-terminal or C-terminal end or internally. The fragment willusually share at least one biological property in common with γ-HRG.γ-HRG fragments will have a consecutive sequence of at least 20, 30, 40,50, or 100 amino acid residues of the NTD of γ-HRG. The preferredfragments have about 30-150 residues which are identical to the sequenceof human γ-HRG in SEQ ID NO:2.

Another type of γ-HRG variant is “chimeric γ-HRG”, which termencompasses a polypeptide comprising full-length γ-HRG or a fragmentthereof fused or bonded to a heterologous polypeptide. The chimera willnormally share at least one biological property in common with γ-HRG.Examples of chimeric γ-HRGs include immunoadhesins and epitope taggedγ-HRG. In another embodiment, the heterologous polypeptide isthioredoxin, a salvage receptor binding epitope, cytotoxic polypeptideor enzyme (e.g., one which converts a prodrug to an active drug).

The term “immunoadhesin” is used interchangeably with the expression“γ-HRG-immunoglobulin chimera” and refers to a chimeric molecule thatcombines a biologically active portion of the γ-HRG with animmunoglobulin sequence. The immunoglobulin sequence preferably, but notnecessarily, is an immunoglobulin constant domain. The immunoglobulinmoiety in the chimeras of the present invention may be obtained fromIgG₁, IgG₂, IgG₃ or IgG₄ subtypes, IgA, IgE, IgD or IgM, but preferablyIgG₁ or IgG₃.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising the entire γ-HRG, or a fragment thereof, fused toa “tag polypeptide”. The tag polypeptide has enough residues to providean epitope against which an antibody thereagainst can be made, yet isshoe enough such that it does not interfere with activity of the γ-HRG.The tag polypeptide preferably is fairly unique so that the antibodythereagainst does not substantially cross-react with other epitopes.Suitable tag polypeptides generally have at least 6 amino acid residuesand usually between about 8-50 amino acid residues (preferably betweenabout 9-30 residues).

As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, orIgG₄) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. I, Y,Pr), chemotherapeutic agents, and toxins such as enzymatically activetoxins of bacterial, fungal, plant or animal origin, or fragmentsthereof. Also, the term includes oncogene product/tyrosine kinaseinhibitors, such as the bicyclic ansamycins disclosed in WO 94/22867;1,2-bis(arylamino) benzoic acid derivatives disclosed in EP 600832;6,7-diamino-phthalazin-1-one derivatives disclosed in EP 600831;4,5-bis(arylamino)-phthalimide derivatives as disclosed in EF 516598; orpeptides which inhibit binding of a tyrosine kinase to a SH2-containingsubstrate protein (see WO 94/07913, for example).

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includeAdriamycin, Doxorubicin, 5-Fluorouracil (5-FU), Cytosine arabinoside(Ara-C), Cyclophosphamide, Thiotepa, Busulfan, Cytoxin, Taxol,Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin, Etoposide,Ifosfamide, Mitomycin C, Mitoxantrone, Vincristine, VP-16, Vinorelbine,Carboplatin, Teniposide, Daunomycin, Caminomycin, Aminopterin,Dactinomycin, Mitomycins, Nicotinamide, Esperamicins (see U.S. Pat. No.4,675,187), Melphalan and other related nitrogen mustards, and endocrinetherapies (such as diethylstilbestrol (DES), Tamoxifen, LHRHantagonizing drugs, progestins, anti-progestins etc).

The term “prodrug” as used in this application refers to a precursor orderivative form of a pharmaceutically active substance that is lesscytotoxic to tumor cells compared to the parent drug and is capable ofbeing enzymatically activated or converted into the more active parentform. The prodrugs of this invention include, but are not limited to,phosphate-containing prodrugs, thiophosphate-containing prodrugs,sulfate-containing prodrugs, peptide-containing prodrugs, D-aminoacid-modified prodrugs, glycosylated prodrugs, β-lactam-containingprodrugs, optionally substituted phenoxyacetamide-containing prodrugs oroptionally substituted phenylacetamide-containing prodrugs,5-fluorocytosine and other 5-fluorouridine prodrugs which can beconverted into the more active cytotoxic free drug. Examples ofcytotoxic drugs that can be derivatized into a prodrug form for use inthis invention include, but are not limited to, those chemotherapeuticagents described above.

“Isolated γ-HRG”, “highly purified γ-HRG” and “substantially homogeneousγ-HRG” are used interchangeably and mean γ-HRG that has been purifiedfrom a γ-HRG source or has been prepared by recombinant or syntheticmethods and is sufficiently free of other peptides or proteins (a) toobtain at least 15 and preferably 20 amino acid residues of theN-terminal or of an internal amino acid sequence by using a spinning cupsequenator or the best commercially available amino acid sequenatormarketed or as modified by published methods as of the filing date ofthis application, or (b) to homogeneity by SDS-PAGE under non-reducingor reducing conditions using Coomassie blue or, preferably, silverstain. Homogeneity here means less than about 5% contamination withother source proteins.

“Biological property” when used in conjunction with “γ-HRG” means havingan effector or antigenic function or activity that is directly orindirectly caused or performed by native sequence γ-HRG of SEQ ID NO:2(whether in its native or denatured conformation).

“Effector functions” include receptor activation (e.g. activation of theErbB2, ErbB3 and/or ErbB4 receptor); enhancement of survival,differentiation and/or proliferation of cells having one or more ofthese receptors (e.g. SK-BR-3 cells, Schwann cells, hepatocytes,glioblastoma cells, epithelial cells, muscle cells, astrocytes and/oroligodendrocytes); receptor binding (e.g. to the ErbB2, ErbB3 and/orErbB4 receptor); mitogenic activity; inducing formation of ion channels(e.g. Na⁺ channel) in a cell membrane; inducing acetylcholine receptorsynthesis at the neuromuscular junction; enhancing formation of asynaptic junction between a neuron and a muscle, nerve or glandularcell; downregulation of estrogen receptor; and cell internalization(possibly associated with nuclear localization). Principle effectorfunctions of native sequence γ-HRG are those demonstrated in the examplebelow, i.e., an ability to bind to the ErbB3 and/or ErbB4 receptor; anability to activate an ErbB receptor, e.g. the ErbB2/ErbB3 and/orErbB2/ErbB4 receptor complexes (as determined in the tyrosinephosphorylation assay); and/or the ability to induce cell proliferation.

An “antigenic function” means possession of an epitope or antigenic sitethat is capable of cross-reacting with antibodies raised against theunique N-terminal domain (NTD) of native sequence γ-HRG, wherein suchantibodies do not significantly cross-react with other known heregulinpolypeptides. The principal antigenic function of a γ-HRG polypeptide isthat it binds with an affinity of at least about 10⁶ L/mole to anantibody which binds specifically to the NTD of γ-HRG. Ordinarily, thepolypeptide binds with an affinity of at least about 10⁷ L/mole.

“Biologically active” when used in conjunction with “γ-HRG” means aγ-HRG polypeptide that exhibits or shares an effector function of nativesequence γ-HRG and that may (but need not) in addition possess anantigenic function.

“Antigenically active” γ-HRG is defined as a polypeptide that possessesan antigenic function of γ-HRG and that may (but need not) in additionpossess an effector function.

“Percent amino acid sequence identity” with respect to the γ-HRGsequence is defined herein as the percentage of amino acid residues inthe candidate sequence that are identical with the residues in the γ-HRGsequence having the deduced amino acid sequence described in FIG. 1,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. None ofN-terminal, C-terminal, or internal extensions, deletions, or insertionsinto the γ-HRG sequence shall be construed as affecting sequenceidentity or homology.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, gastric cancer,pancreatic cancer, glial cell tumors such as glioblastoma andneurofibromatosis, cervical cancer, ovarian cancer, liver cancer,bladder cancer, hepatoma, breast cancer, colon cancer, colorectalcancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer,renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer.

“Determining disease status” refers to the act of determining likelihoodof patient survival and time to relapse for neoplastic diseases,particularly breast, ovarian, stomach, endometrial, salivary gland,lung, kidney, colon, and bladder carcinomas. In particular, γ-HRG can beused to quantify erbB (e.g., erbB2, erbB3 or erbB4, but normally erbB2)overexpression in cancerous tissue taken from a patient suffering fromcarcinoma. This can also be referred to as “determining the propercourse of treatment for patients suffering from cancer”. For example,those patients characterized by erbB2 overexpression may require moreaggressive treatment (e.g. chemo- or radiotherapy treatment) than mightotherwise be indicated by other diagnostic factors. This phraseencompasses diagnosing patients suffering from high grade ductalcarcinoma in situ. See, e.g., Disis et al., Cancer Research, 54:16-20(1994).

The word “sample” refers to tissue, body fluid, or a cell from apatient. Normally, the tissue or cell will be removed from the patient,but in vivo diagnosis is also contemplated. In the case of a solidtumor, a tissue sample can be taken from a surgically removed tumor andprepared for testing by conventional techniques. In the case oflymphomas and leukemias, lymphocytes, leukemic cells, or lymph tissueswill be obtained and appropriately prepared. Other patient samples,including urine, tear drops, serum, cerebrospinal fluid, feces, sputum,cell extracts etc will also be useful for particular tumors.

The expression “labelled” when used herein refers to a molecule (e.g.γ-HRG or anti-γ-HRG antibody) which has been conjugated, directly orindirectly, with a detectable compound or composition. The label may bedetectable by itself (e.g. radioisotope labels or fluorescent labels)or, in the case of an enzymatic label, may catalyze a chemicalalteration of a substrate compound or composition which is detectable.

By “solid phase” is meant a non-aqueous matrix to which a reagent ofinterest (e.g. γ-HRG or an antibody thereto) can adhere. Examples ofsolid phases encompassed herein include those formed partially orentirely of glass (e.g., controlled pore glass), polysaccharides (e.g.,agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones.In certain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g., an affinity chromatography column). This term alsoincludes a discontinuous solid phase of discrete particles, such asthose described in U.S. Pat. No. 4,275,149.

The phrase “activating an ErbB receptor’ refers to the act of causingthe intracellular kinase domain of an ErbB receptor to phosphorylatetyrosine residues. Generally, this will involve binding of γ-HRG to anreceptor complex of two or more ErbB receptors (e.g., an ErbB2/ErbB3 orErbB2/ErbB4 complex) which activates a kinase domain of one or more ofthose receptors and thereby results in phosphorylation of tyrosineresidues in one or more of the receptors, and/or phosphorylation oftyrosine residues in additional substrate polypeptides(s). ErbB receptorphosphorylation can be quantified using the tyrosine phosphorylationassays described below.

The expression “enhancing survival of a cell” refers to the act ofincreasing the period of existence of a cell, relative to an untreatedcell which has not been exposed to γ-HRG, either in vitro or in vivo.

The phrase “enhancing proliferation of a cell” encompasses the step ofincreasing the extent of growth and/or reproduction of the cell,relative to an untreated cell, either in vitro or in vivo. An increasein cell proliferation in cell culture can be detected by counting thenumber of cells before and after exposure to γ-HRG (see the Examplebelow). The extent of proliferation can be quantified via microscopicexamination of the degree of confluency. Cell proliferation can also bequantified by measuring ³H uptake by the cells.

By “enhancing differentiation of a cell” is meant the act of increasingthe extent of the acquisition or possession of one or morecharacteristics or functions which differ from that of the original cell(i.e. cell specialization). This can be detected by screening for achange in the phenotype of the cell (e.g. identifying morphologicalchanges in the cell).

A “glial cell” is derived from the central and peripheral nervous systemand can be selected from oligodendroglial, astrocyte, ependymal, ormicroglial cells as well as satellite cells of ganglia and theneurolemmal or Schwann cells around peripheral nerve fibers.

“Muscle cells” include skeletal, cardiac or smooth muscle tissue cells.This term encompasses those cells which differentiate to form morespecialized muscle cells (e.g. myoblasts).

“Isolated γ-HRG nucleic acid” is RNA or DNA free from at least onecontaminating source nucleic acid with which it is normally associatedin the natural source and preferably substantially free of any othermammalian RNA or DNA. The phrase “free from at least one contaminatingsource nucleic acid with which it is normally associated” includes thecase where the nucleic acid is present in the source or natural cell butis in a different chromosomal location or is otherwise flanked bynucleic acid sequences not normally found in the source cell. An exampleof isolated γ-HRG nucleic acid is RNA or DNA that encodes a biologicallyactive γ-HRG sharing at least 75%, more preferably at least 80%, stillmore preferably at least 85%, even more preferably 90%, and mostpreferably 95% sequence identity with the human γ-HRG shown in FIG. 1.

“Stringent conditions” are those that (a) employ low ionic strength andhigh temperature for washing, for example, 0.015 M NaCl/0.0015 M sodiumcitrate/0.1% NaDodSO₄ (SDS) at 50° C., or (b) employ duringhybridization a denaturing agent such as formamide, for example, 50%(vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMNaCl, 75 mM sodium citrate at 42° C. Another example is use of 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 μg/mL), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC and 0.1% SDS.

“Moderately stringent conditions” are described in Sambrook et al.,Molecular Cloning: A Laboratory Manual (New York: Cold Spring HarborLaboratory Press, 1989), and include the use of a washing solution andhybridization conditions (e.g., temperature, ionic strength, and % SDS)less stringent than described above. An example of moderately stringentconditions is a condition such as overnight incubation at 37° C. in asolution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10%dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA,followed by washing the filters in 1×SSC at about 37-50° C. The skilledartisan will recognize how to adjust the temperature, ionic strength,etc., as necessary to accommodate factors such as probe length and thelike.

The expression “control sequences” refers to DNA sequences necessary forthe expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

A γ-HRG “antagonist” is a molecule which prevents, or interferes with, aγ-HRG effector function (e.g. a molecule which prevents or interfereswith binding and/or activation of an ErbB receptor by γ-HRG). Suchmolecules can be screened for their ability to competitively inhibitErbB receptor activation by γ-HRG in the tyrosine phosphorylation assaydisclosed herein, for example. Preferred antagonists are those which donot substantially interfere with the interaction of other heregulinpolypeptides with ErbB receptor(s). Examples of γ-HRG antagonistsinclude neutralizing antibodies against γ-HRG and antisensepolynucleotides against the γ-HRG gene.

The terms “antisense oligodeoxynucleotide” and “antisense oligo” referto a polynucleotide which hybridizes with an area of γ-HRG mRNA or DNAand thereby prevents or reduces production of γ-HRG polypeptide in vitroor in vivo. Preferred antisense polynucleotides are those which arecomplementary to at least a portion of the γ-HRG NTD-coding region ofFIG. 1. This term encompasses “modified” polynucleotides, examples ofwhich are described herein.

The term “antibody” is used in the broadest sense and specificallycovers single anti-γ-HRG monoclonal antibodies and anti-γ-HRG antibodycompositions with polyepitopic specificity (including neutralizing andnon-neutralizing antibodies). The antibody of particular interest hereinis one which does not significantly cross-react with other knownheregulin proteins, such as those described in the background sectionabove and thus is one which “binds specifically” to γ-HRG. Hence,antibodies which bind to the unique NTD of γ-HRG may be particularlyuseful. In such embodiments, the extent of binding of the antibody tonon-γ-HRG proteins will be less than 10% as determined byradioimmunoprecipitation (RIA), for example.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally-occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen.

The monoclonal antibodies herein include hybrid and recombinantantibodies produced by splicing a variable (including hypervariable)domain of an anti-γ-HRG antibody with a constant domain (e.g.“humanized” antibodies), or a light chain with a heavy chain, or a chainfrom one species with a chain from another species, or fusions withheterologous proteins, regardless of species of origin or immunoglobulinclass or subclass designation, as well as antibody fragments (e.g., Fab,F(ab)₂, and Fv), so long as they exhibit the desired biologicalactivity. (See, e.g., U.S. Pat. No. 4,816,567 and Mage & Lamoyi, inMonoclonal Antibody Production Techniques and Applications, pp. 79-97(Marcel Dekker, Inc.), New York (1987)).

Thus, the modifier “monoclonal” indicates the character of the antibodyas being obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler & Milstein,Nature 256:495 (1975), or may be made by recombinant DNA methods (U.S.Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolatedfrom phage libraries generated using the techniques described inMcCafferty et al., Nature 348:552-554 (1990), for example.

“Humanized” forms of non-human (e.g. murine) antibodies are specificchimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab)₂ of other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulin (recipient antibody) in which residues from thecomplementarity determining regions (CDRs) of the recipient antibody arereplaced by residues from the CDRs of a non-human species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human FR residues. Furthermore, the humanized antibody may compriseresidues which are found neither in the recipient antibody nor in theimported CDR or FR sequences. These modifications are made to furtherrefine and optimize antibody performance. In general, the humanizedantibody will comprise substantially all of at least one, and typicallytwo, variable domains, in which all or substantially all of the CDRregions correspond to those of a non-human immunoglobulin and all orsubstantially all of the FR residues are those of a human immunoglobulinconsensus sequence. The humanized antibody optimally also will compriseat least a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin.

By “neutralizing antibody” is meant an antibody molecule as hereindefined which is able to block or significantly reduce an effectorfunction of native sequence γ-HRG. For example, a neutralizing antibodymay inhibit or reduce the ability of γ-HRG to activate an ErbB receptorin the tyrosine phosphorylation assay described herein. The neutralizingantibody may also block the mitogenic activity of γ-HRG in the cellproliferation assay disclosed herein.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those prone to have the disorder or thosein which the disorder is to be prevented.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as sheep, dogs, horses, cats, cows, etc. Preferably,the mammal herein is human.

“Pharmaceutically acceptable” carriers, excipients, or stabilizers areones which are nontoxic to the cell or mammal being exposed thereto atthe dosages and concentrations employed. Often the physiologicallyacceptable carrier is an aqueous pH buffered solution. Examples ofphysiologically acceptable carriers include buffers such as phosphate,citrate, and other organic acids; antioxidants including ascorbic acid;low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as Tween™, polyethylene glycol (PEG), and Pluronics™.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drugto a mammal. The components of the liposome are commonly arranged in abilayer formation, similar to the lipid arrangement of biologicalmembranes.

II. Modes for Practicing the Invention

1. γ-HRG Polypeptide & Nucleic Acid

The DNA and amino acid sequences of human γ-HRG are depicted in FIG. 1.It is contemplated that the novel γ-HRG described herein may be a memberof a family of growth factors having suitable sequence identity thattheir DNA may hybridize to the DNA in the unique N-terminal domain (NTD)of FIG. 1 (or fragments thereof) under moderately stringent to stringentconditions. An example of such γ-HRG variant is the isoform shown inFIG. 5. Thus, a further aspect of this invention includes DNA whichhybridizes under moderately stringent to stringent conditions with theDNA encoding the NTD of γ-HRG. Techniques for isolating such nativesequence γ-HRG molecules and making variant γ-HRG follow.

Techniques suitable for the production of γ-HRG are well known in theart and include isolating γ-HRG from an endogenous source of thepolypeptide, peptide synthesis (using a peptide synthesizer) andrecombinant techniques (or any combination of these techniques). Thepreferred technique for production of γ-HRG is a recombinant techniqueto be described below. See also U.S. Pat. No. 5,364,934 with respect tovectors, host cells, etc for the recombinant production of γ-HRG.

To produce γ-HRG polypeptide, the DNA encoding γ-HRG is isolated (e.g.from a cDNA library as disclosed in Example 1 below) and operably linkedto other nucleic acid in a suitable vector. The DNA thus isolated may bemutated so as to enhance expression depending on the expression systemselected. For example, nucleotide substitutions may be made which avoid5′ stem and loop structures in the transcribed mRNA and/or to providecodons that are more readily transcribed by the selected host (e.g. thewell-known preference codons for E. coli or yeast expression). Thevector is a plasmid or other DNA that is capable of replicating within ahost cell and can be used for cloning (i.e. to produce usable quantitiesof the nucleic acid) and/or to direct expression of γ-HRG. Vector designdepends, among other things, on the intended use and host cell for thevector. The vector components generally include, but are not limited to,one or more of the following: an N-terminal signal sequence, an originof replication, one or more marker genes, an enhancer element, apromoter, an operator sequence, a ribosome binding site, and atranscription termination sequence.

A particularly useful plasmid for mammalian cell culture production ofγ-HRG is pRK5 (EP 307,247) and derivatives thereof, or pSV16B (WO91/08291 published 13 Jun. 1991). Another useful vector is disclosed inWO 96/04391. A host cell is generally transformed with the vector.Suitable host cells for cloning or expressing the vectors herein areprokaryote host cells (such as E. coli, strains of Bacillus, Pseudomonasand other bacteria), yeast and other eukaryotic microbes, and highereukaryote cells (such as Chinese hamster ovary (CHO) cells and othermammalian cells). The cells may also be present in live animals (forexample, in cows, goats or sheep). Insect cells may also be used.Cloning and expression methodologies are well known in the art. Toobtain expression of γ-HRG, an expression vector is introduced into hostcells by transformation or transfection and the resulting recombinanthost cells are cultured in conventional nutrient media, modified asappropriate for inducing promoters, selecting recombinant cells, oramplifying γ-HRG DNA. In general, principles, protocols, and practicaltechniques for maximizing the productivity of in vitro mammalian cellcultures can be found in Mammalian Cell Biotechnology: a PracticalApproach, M. Butler, ed. (IRL Press, 1991).

The terms “transformation” and “transfection” are used interchangeablyherein and refer to the process of introducing DNA into a cell.Following transformation or transfection, the γ-HRG DNA may integrateinto the host cell genome, or may exist as an extrachromosomal element.If prokaryotic cells or cells that contain substantial cell wallconstructions are used as hosts, the preferred methods of transfectionof the cells with DNA is the calcium treatment method described by Cohenet al., Proc. Natl. Acad. Sci. U.S.A., 69:2110-2114 (1972) or thepolyethylene glycol method of Chung et al., Nuc. Acids. Res. 16:3580(1988). If yeast are used as the host, transfection is generallyaccomplished using polyethylene glycol, as taught by Hinnen, Proc. Natl.Acad. Sci. U.S.A., 75:1929-1933 (1978). If mammalian cells are used ashost cells, transfection generally is carried out by the calciumphosphate precipitation method, Graham et al., Virology 52:546 (1978),Gorman et al., DNA and Protein Eng. Tech. 2:3-10 (1990). However, otherknown methods for introducing DNA into prokaryotic and eukaryotic cells,such as nuclear injection, electroporation, or protoplast fusion alsoare suitable for use in this invention.

Particularly useful in this invention are expression vectors thatprovide for the transient expression in mammalian cells of DNA encodingγ-HRG. In general, transient expression involves the use of anexpression vector that is able to efficiently replicate in a host cell,such that the host cell accumulates many copies of the expression vectorand, in turn, synthesizes high levels of a desired polypeptide encodedby the expression vector. Transient expression systems, comprising asuitable expression vector and a host cell, allow for the convenientpositive identification of polypeptides encoded by cloned DNAs, as wellas for the rapid screening of such polypeptides for desired biologicalor physiological properties.

It is further envisioned that the γ-HRG of this invention may beproduced by homologous recombination, as provided for in WO 91/06667,published 16 May 1991. Briefly, this method involves transforming a cellcontaining an endogenous-HRG gene with a homologous DNA, whichhomologous DNA comprises (a) an amplifiable gene (e.g. a gene encodingdihydrofolate reductase (DHFR)), and (b) at least one flanking sequence,having a length of at least about 150 base pairs, which is homologouswith a nucleotide sequence in the cell genome that is within or inproximity to the gene encoding γ-HRG. The transformation is carried outunder conditions such that the homologous DNA integrates into the cellgenome by recombination. Cells having integrated the homologous DNA arethen subjected to conditions which select for amplification of theamplifiable gene, whereby the γ-HRG gene is amplified concomitantly. Theresulting cells are then screened for production of desired amounts ofγ-HRG. Flanking sequences that are in proximity to a gene encoding γ-HRGare readily identified, for example, by the method of genomic walking,using as a starting point the nucleotide sequence of γ-HRG of FIG. 1.

γ-HRG preferably is recovered from the culture medium as a secretedpolypeptide, although it also may be recovered from host cell lysates.As a first step, the particulate debris, either host cells or lysedfragments, is removed, for example, by centrifugation orultrafiltration; optionally, the protein may be concentrated with acommercially available protein concentration filter, followed byseparating the γ-HRG from other impurities by one or more purificationprocedures selected from: fractionation on an immunoaffinity column;fractionation on an ion-exchange column; ammonium sulphate or ethanolprecipitation; reverse phase HPLC; chromatography on silica;chromatography on heparin Sepharose; chromatography on a cation exchangeresin; chromatofocusing; SDS-PAGE; and gel filtration (e.g. using a HighLoad Superdex™ 75 prep grade column, see Example below). Where the γ-HRGis expressed initially as an insoluble, aggregated form (especially inbacterial host cells), it may be necessary to solubilize and renaturethe γ-HRG using techniques available in the art for solubilizing andrenaturing recombinant protein refractile bodies (see, e.g., U.S. Pat.No. 4,511,502).

γ-HRG variants (see below) are recovered in the same fashion as nativesequence γ-HRG, taking account of any substantial changes in propertiesoccasioned by the variation. For example, preparation of epitope taggedγ-HRG, facilitates purification using an immunoaffinity columncontaining antibody to the epitope to adsorb the fusion polypeptide.Immunoaffinity columns such as a rabbit polyclonal anti-γ-HRG column canbe employed to absorb the γ-HRG variant by binding it to at least oneremaining immune epitope.

Amino acid sequence variants of native sequence γ-HRG are prepared byintroducing appropriate nucleotide changes into the native sequencer-HRGDNA, or by in vitro synthesis of the desired γ-HRG polypeptide. Suchvariants include, for example, deletions from, or insertions orsubstitutions of, residues in the amino acid sequence shown for humanγ-HRG in FIG. 1. The amino acid changes also may alterpost-translational processes of the native sequence γ-HRG, such aschanging the number or position of N- and/or O-linked glycosylationsites. Potential N-linked glycosylation sites are shown in FIG. 1.Generally, the Asn residue will be replaced with a Gln, but othersubstitutions or deletions are possible.

A useful method for identification of certain residues or regions of thenative γ-HRG polypeptide that are preferred locations for mutagenesis is“alanine scanning mutagenesis” as described by Cunningham and Wells,Science 244:1081-1085 (1989).

Amino acid sequence deletions generally range from about 1 to 30residues, more preferably about 1 to 10 residues, and typically arecontiguous. Contiguous deletions ordinarily are made in even numbers ofresidues, but single or odd numbers of deletions are within the scopehereof. Deletions may be introduced into regions of low homology amongvarious mammalian γ-HRGs to modify the activity of γ-HRG. Deletions fromγ-HRG in the EGF-like domain will be more likely to modify thebiological activity of γ-HRG more significantly. An exemplary γ-HRGdeletion mutant is γ-HRG with residues 749-768 in FIG. 1 deleted. Otherexemplary deletions include native sequence γ-HRG with one or more ofthe N-linked glycosylation sites identified in FIG. 1 deleted and/or anyone or more of the residues associated with the potential proteasecleavage sites removed (i.e. any one or more of residues 411-414,440-441, 481-482, 500-501, 606-607 removed) and/or with any one or morecysteine residues removed. Regions of interest in the γ-HRG amino acidsequence of FIG. 1 include residues 1-748 (i.e. γ-HRG lacking the β3C-terminal domain); 1-703 (i.e. γ-HRG lacking the EGF-like domain);1-569 (i.e. γ-HRG lacking the EGF-like domain and spacer domain); 1-560(i.e. γ-HRG lacking the EGF-like domain, spacer domain and 1 g domain);342-363 (the hydrophobic region); 364-560; 364-768; 411-560; 411-768;412-560; 412-768; 413-560; 413-768; 414-560; 414-768; 415-560; 415-768;441-560; 441-768; 482-560; 482-768; 501-560 501-768; 607-560; 607-768.These regions can be used in the production of γ-HRG polypeptides whichconsist essentially of, or comprise, these regions. Such deletionmutants may further comprise additional internal deletions and/orsubstitutions and/or may be fused to a heterologous polypeptide, e.g.,an immunogenic polypeptide, for use in generating anti-γ-HRG antibodies.These mutants may also comprise an additional carboxyl or amino-terminalamino acid residue (e.g. an amino-terminal methionyl residue).

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Intrasequence insertions (i.e.,insertions within the mature γ-HRG sequence) may range generally fromabout 1 to 10 residues, more preferably 1 to 5, most preferably 1 to 3.Insertions are preferably made in even numbers of residues, but this isnot required. Examples of terminal insertions include γ-HRG with anN-terminal methionyl residue, an artifact of the direct production ofγ-HRG in recombinant cell culture.

A preferred type of insertion variant is chimeric γ-HRG. Fusion proteinscomprising γ-HRG linked to a heterologous polypeptide can be constructedusing recombinant DNA techniques, or the heterologous polypeptide can becovalently bound to the γ-HRG polypeptide by techniques well known inthe art such as the use of the heterobifunctional crosslinking reagents.Exemplary coupling agents include N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives ofimidoesters (such as dimethyl adipimidate HCL), active esters (such asdisuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azidocompounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazoniumderivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),diisocyanates (such as tolyene 2,6-diisocyanate), and bis-activefluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

Chimeric γ-HRG polypeptides include fusions of γ-HRG with immunogenicpolypeptides, e.g., bacterial polypeptides such asp-lactamase or anenzyme encoded by the E. coli trp locus, or yeast protein, and fusionswith proteins such as albumin, or ferritin, as described in WO 89/02922published 6 Apr. 1989. Another example of chimeric γ-HRG is thethioredoxin fusion protein described in the Example herein.

In one embodiment, the chimeric polypeptide comprises a fusion of theγ-HRG (or a fragment thereof) with a tag polypeptide which provides anepitope to which an anti-tag antibody can selectively bind. The epitopetag is generally proved at the amino- or carboxyl-terminus of the γ-HRG.Such epitope tagged forms of the γ-HRG are desirable, as the presencethereof can be detected using a labelled antibody against the tagpolypeptide. Also, provision of the epitope tag enables the γ-HRG to bereadily purified by affinity purification using the anti-tag antibody.Tag polypeptides and their respective antibodies are well known in theart. Examples include the flu HA tag polypeptide and its antibody 12CA5,(Field et al., Mol. Cell. Biol. 8:2159-2165 (1988)); the c-myc tag andthe 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et al.,Molecular and Cellular Biology 5(12):3610-3616 (1985)); and the HerpesSimplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al.,Protein Engineering 3(6):547-553 (1990)).

The chimeric γ-HRG may also comprise an immunoadhesin having a longerhalf-life than native γ-HRG. Immunoadhesins constructed from apolypeptide linked to a heterologous immunoglobulin constant domainsequence are known in the art.

The simplest and most straightforward immunoadhesin design combinesγ-HRG with the hinge and Fc regions of an immunoglobulin heavy chain.Ordinarily, when preparing the γ-HRG-immunoglobulin chimeras of thepresent invention, nucleic acid encoding the γ-HRG, or a fragmentthereof, will be fused C-terminally to nucleic acid encoding theN-terminus of an immunoglobulin constant domain sequence, howeverN-terminal fusions are also possible. Typically, in such fusions theencoded chimeric polypeptide will retain at least functionally activehinge, CH2 and CH3 domains of the constant region of an immunoglobulinheavy chain. Fusions are also made to the C-terminus of the Fc portionof a constant domain, or immediately N-terminal to the CH1of the heavychain or the corresponding region of the light chain. Chimeric γ-HRG ismost conveniently constructed by fusing the cDNA sequence encoding theγ-HRG portion in-frame to the tag polypeptide or immunoglobulin DNAsequence, for example, and expressing the resultant DNA fusion constructin appropriate host cells.

Another type of chimeric γ-HRG which is encompassed by the presentinvention is γ-HRG fused with a cytotoxic polypeptide (e.g. anenzymatically active toxin of bacterial, fungal, plant or animal origin,or fragments thereof). Alternatively, the toxin may be covalentlyattached to isolated γ-HRG. Enzymatically active toxins and fragmentsthereof which can be used include diphtheria A chain, nonbinding activefragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A (e.g. ricin A chain), abrin A chain, modeccin Achain, alpha-sarcin Aleurites fordii proteins, dianthin proteins,Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordicacharantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor,gelonin, mitogellin restrictocin, phenomycin, enomycin and thetricothecenes.

A still further type of chimeric γ-HRG, is γ-HRG polypeptide fused witha prodrug-activating enzyme which converts a prodrug (e.g. a peptidylchemotherapeutic agent, see WO81/01145) to an active anti-cancer drug.See, for example, WO 88/07378 and U.S. Pat. No. 4,975,278. ThE enzymecomponent of such a chimeric molecule includes any enzyme capable ofacting on a prodrug in such a way so as to covert it into its moreactive, cytotoxic form.

Enzymes that are useful in the method of this invention include, but arenot limited to, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug, 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs; D-alanylcarboxypeptidases, useful forconverting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidaseuseful for converting glycosylated prodrugs into free drugs; β-lactamaseuseful for converting drugs derivatized with β-lactams into free drugs;and penicillin amidases, such as penicillin V amidase or penicillin Gamidase, useful for converting drugs derivatized at their aminenitrogens with phenoxyacetyl or phenylacetyl groups, respectively, intofree drugs. Alternatively, antibodies with enzymatic activity, alsoknown in the art as “abzymes”, can be used to convert the prodrugs ofthe invention into free active drugs (see, e.g., Massey, Nature328:457-458 (1987)).

Yet a further type of chimeric polypeptide is γ-HRG fused to a salvagereceptor binding epitope. Such chimeric molecules may have enhancedserum half-lives when compared to native sequence γ-HRG. The phrase“long half-life” as used in connection with γ-HRG derivatives, concernsγ-HRG derivatives having a longer plasma half-life and/or slowerclearance than a corresponding native sequence γ-HRG.

A systematic method for preparing such a chimeric polypeptide having anincreased in vivo half-life comprises several steps. The first involvesidentifying the sequence and conformation of a salvage receptor bindingepitope of an Fc region of an IgG molecule. Once this epitope isidentified, the sequence of the γ-HRG is modified to include thesequence and conformation of the identified binding epitope. After thesequence is mutated, the γ-HRG variant is tested to see if it has alonger in vivo half-life than that of the original molecule. If theγ-HRG variant does not have a longer in vivo half-life upon testing, itssequence is further altered to include the sequence and conformation ofthe identified binding epitope. The altered γ-HRG is tested for longerin vivo half-life, and this process is continued until a molecule isobtained that exhibits a longer in vivo half-life.

The salvage receptor binding epitope generally constitutes a regionwherein any one or more amino acid residues from one or two loops of aFc domain are transferred to the γ-HRG. Even more preferably, three ormore residues from one or two loops of the Fc domain are transferred.Still more preferred, the epitope is taken from the CH2 domain of the Fcregion (e.g., of an IgG).

In one most preferred embodiment, the salvage receptor binding epitopecomprises the sequence (5′ to 3′): PKNSSMISNTP (SEQ ID NO:5), andoptionally further comprises a sequence selected from the groupconsisting of HQSLGTQ (SEQ ID NO:6), HQNLSDGK (SEQ ID NO:7), HQNISDGK(SEQ ID NO:8), or VISSHLGQ (SEQ ID NO:9). In another most preferredembodiment, the salvage receptor binding epitope is a polypeptidecontaining the sequence(s) (5′ to 3′): HQNLSDGK (SEQ ID NO:7), HQNISDGK(SEQ ID NO:8), or VISSHLGQ (SEQ ID NO:9) and the sequence: PKNSSMISNTP(SEQ ID NO:5).

Alternatively, γ-HRG may be fused to a molecule (such as an antibody)which binds to a triggering molecule on a leukocyte such as a T-cellreceptor molecule (e.g. CD2 or CD3), or Fc receptors for IgG (FcγR),such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focuscellular defense mechanisms to an erbB-expressing cell. Similarly,chimeric γ-HRG polypeptides are contemplated herein which localizecytotoxic agents to cells which express an erbB gene. For example, theheterologous polypeptide in such a chimeric γ-HRG polypeptide may be onewhich binds the cytotoxic agent (e.g. antibodies directed againstsaporin, anti-interferon-α, vinca alkaloid, ricin A chain, methotrexateor radioactive isotope hapten).

A third group of variants are amino acid substitution variants. Thesevariants have at least one amino acid residue in the native sequenceγ-HRG molecule removed and a different residue inserted in its place.For example, the substitution variant may be one which differs from theamino acid sequence shown in FIG. 1 for mature γ-HRG by the substitutionof one amino acid for another at one, two, three or more positionswithin the FIG. 1 amino acid sequence.

Variations in the native sequence as described above can be made usingany of the techniques and guidelines for conservative andnon-conservative mutations set forth in U.S. Pat. No. 5,364,934. Seeespecially Table 1 therein and the discussion surrounding this table forguidance on selecting amino acids to change, add, or delete.

Any cysteine residues not involved in maintaining the properconformation of native γ-HRG also may be substituted, generally withserine, to improve the oxidative stability of the molecule and preventaberrant crosslinking. Also, residues in potential protease cleavagesites (i.e. any one or more of residues 411-414, 440-441, 481-482,500-501, 606-607) may be replaced by other residues.

Representative substitutions include γ-HRG with the β-type EGF-likedomain substituted with an α-type EGF-like domain, and γ-HRG with theβ-type EGF-like domain replaced with the β-type EGF-like domain of ratNDF or ARIA. Further exemplary substitutions of human γ-HRG of FIG. 1include any one or more of the following substitutions: hγ-HRG(Cys297-Ser), hγ-HRG (Cys639-Ser), hγ-HRG (Pro90-Ala), hγ-HRG (His159-Arg), hγ-HRG (Glu237-Asp), hγ-HRG (Asn329-Gln), hγ-HRG (Leu365-Val),hγ-HRG (Val396-Leu), hγ-HRG (Gln455-Asn), hγ-HRG (Val468-Leu), hγ-HRG(Thr520-Ser), hγ-HRG (Lys 570-Arg), hγ-HRG (Leu 593-Ala), hγ-HRG(Val657-Ala), hγ-HRG (Asn467-Gln), hγ-HRG (Asn 691-Gln).

Nucleic acid molecules encoding amino acid sequence variants of nativesequence γ-HRG are prepared by a variety of methods known in the art.These methods include, but are not limited to, isolation from a naturalsource (in the case of naturally occurring amino acid sequence variants)or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared variant or a non-variant version of native sequence γ-HRG.

Covalent modifications of γ-HRG polypeptides are included within thescope of this invention. Both native sequence γ-HRG and amino acidsequence variants thereof may be covalently modified. Covalentmodifications of γ-HRG or fragments thereof may be introduced into themolecule by reacting targeted amino acid residues of the γ-HRG orfragments thereof with an organic derivatizing agent that is capable ofreacting with selected side chains or the N- or C-terminal residues. SeeU.S. Pat. No. 5,364,934 concerning potential covalent modifications ofγ-HRG.

For tumor targeting, it may be beneficial to covalently conjugate γ-HRGwith a cytotoxic agent, such as those described above. For example, avariety of radionuclides (e.g. ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y and ¹⁸⁶Re) areavailable for the production of radioconjugated γ-HRG. Carbon-14-labeled1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid(MX-DTPA) is an exemplary chelating agent for conjugation ofradionucleotide to the γ-HRG. See WO94/11026.

Another type of covalent modification of γ-HRG comprises linking theγ-HRG polypeptide to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol, polypropylene glycol, polyoxyalkylenes, orcopolymers of polyethylene glycol and polypropylene glycol. γ-HRG alsomay be entrapped in microcapsules prepared, for example, by coacervationtechniques or by interfacia polymerization (for example,hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively), in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, Oslo, A., Ed., (1980).

Once amino acid sequence variants and covalent variants have been made,it is routine to screen for those molecules which are biologicallyand/or antigenically active. Competitive-type immunoassays can beemployed for determining whether the variant is able to cross-react withantibodies raised against native sequence γ-HRG. Other potentialmodifications of protein or polypeptide properties such as redox orthermal stability, hydrophobicity, susceptibility to proteolyticdegradation, or the tendency to aggregate with carriers or intomultimers are assayed by methods well known in the art.

Generally, the variants of interest will have any one or more of thefollowing properties: (a) the ability to bind to ErbB3 and/or ErbB4; (b)the ability to activate ErbB receptor(s) in ErbB2/ErbB3 and/orErbB2/ErbB4 receptor complexes; and (c) the ability to stimulateproliferation of cells which express the ErbB2 and ErbB3 receptor and/orthe ErbB2 and ErbB4 receptor.

To screen for property (a), the ability of the γ-HRG variant to bind toeither or both the ErbB3 and ErbB4 receptor can be readily determined invitro. For example, immunoadhesin forms of these receptors can begenerated (see below) and the ErbB3 or ErbB4 immunoadhesin can beimmobilized on a solid phase (e.g. on assay plates coated withgoat-anti-human antibody). The ability of γ-HRG to bind to theimmobilized immunoadhesin can then be determined, e.g. by determiningcompetitive displacement by other heregulin molecules. For more details,see the ¹²⁵I-HRG binding assay described in the Example below.

As to property (b), the tyrosine phosphorylation assay using MCF7 cellsdescribed in the Example provides a means for screening for activationof ErbB receptors. In an alternative embodiment of the invention, theKIRA-ELISA described in WO 95/14930 can be used to qualitatively andquantitatively measure the ability of a γ-HRG variant to activate anErbB receptor. Briefly, according to the assay described in thisapplication, MCF7 cells (which produce measurable levels of ErbB2, ErbB3and ErbB4) at cell densities of 60% to 75% confluency are added to eachwell in a flat-bottom-96 well culture plate and cultured overnight at37° C. in 5% CO². The following morning the well supernatants aredecanted, and the plates are lightly tamped on a paper towel. Mediacontaining either culture medium (control), native sequence γ-HRG orvariant γ-HRG is then added to each well. The cells are stimulated at37° C. for about 30 min., the well supernatants are decanted, and theplates are once again lightly tamped on a paper towel. To lyse the cellsand solubilize the receptors, 100 μl of lysis buffer is added to eachwell. Lysis buffer consists of 150 mM NaCl containing 50 mM HEPES(Gibco), 0.5% Triton-X 100 (Gibco), 0.01% thimerosal, 30 KIU/mlaprotinin (ICN Biochemicals, Aurora, Ohio), 1 mM4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF; ICNBiochemicals), 50 μM leupeptin (ICN Biochemicals), and 2 mM sodiumorthovanadate (Na₃VO₄, Sigma Chemical Co, St. Louis, Mo.), pH 7.5. Theplate is then agitated gently on a plate shaker (Bellco Instruments,Vineland, N.J.) for 60 min. at room temperature.

While the cells are being solubilized, an ELISA microtiter plate (NuncMaxisorp, Inter Med, Denmark) coated overnight at 4° C. with anaffinity-purified polyclonal antibody directed against the ErbB2, ErbB3or ErbB4 extracellular domain (depending on the receptor of interest) isdecanted, tamped on a paper towel and blocked with 150 μl/well of BlockBuffer (PBS containing 0.5% BSA (Intergen Company, Purchase, N.Y.) and0.01% thimerosal) for 60 min. at room temperature with gentle agitation.After 60 minutes, the anti-ErbB coated plate is washed 6 times with washbuffer (PBS containing 0.05% Tween-20 and 0.01% thimerosal) using anautomated plate washer (ScanWasher 300, Skatron Instruments, Inc,Sterling, Va.).

The lysate containing solubilized ErbB receptor from the cell-culturemicrotiter well is transferred (85 μl/well) to an anti-ErbB coated andblocked ELISA well and is incubated for 2 h at room temperature withgentle agitation. The unbound receptor is removed by washing with washbuffer and 100 μl of biotinylated 4G10 (anti-phosphotyrosine antibody)in dilution buffer (PBS containing 0.5% BSA, 0.05% Tween-20, 5 mM EDTA,and 0.01% thimerosal), is added to each well. After incubation for 2 hat room temperature the plate is washed and 100 μl of HRPO-conjugatedstreptavidin (Zymed Laboratories, S. San Francisco, Calif.) in dilutionbuffer is added to each well. The plate is incubated for 30 minutes atroom temperature with gentle agitation. The free avidin-conjugate iswashed away and 100 μl freshly prepared substrate solution (tetramethybenzidine (TMB); 2-component substrate kit; Kirkegaard and Perry,Gaithersburg, Md.) is added to each well. The reaction is allowed toproceed for 10 minutes, after which the color development is stopped bythe addition of 100 μl/well 1.0 M H₃PO₄. The absorbance at 450 nm isread with a reference wavelength of 650 nm (ABS_(450/650)), using a vmaxplate reader (Molecular Devices, Palo Alto, Calif.) controlled with aMacintosh Centris 650 (Apple Computers, Cupertino, Calif.) and DeltaSoftsoftware (BioMetallics, Inc, Princeton, N.J.).

Thus, the degree of ErbB receptor phosphorylation induced by the variantγ-HRG can be compared to that induced by native sequence γ-HRG, as wellas the control (presumably no activation).

Finally with respect to property (c), the ability of the γ-HRG variantto stimulate proliferation of a cell which expresses the ErbB2 and ErbB3receptor and/or ErbB2 and ErbB4 receptor can readily be determined incell culture. Useful cells for this experiment include MCF7 and SK-BR-3cells obtainable from the ATCC. These tumor cell lines may be plated incell culture plates and allowed to adhere thereto. The γ-HRG variant andnative sequence γ-HRG control may be added at a final concentration of,e.g., 1 nM. Monolayers may be washed and stained/fixed with crystalviolet. Proliferation can therefore be quantified as described. See theExample below for more details. Another useful cell for determiningproliferation capacity of γ-HRG, including variants thereof, is theSchwann cell. See Li et al., supra.

2. Therapeutic Compositions and Methods

γ-HRG is also believed to be useful in promoting the development,maintenance, and/or regeneration of neurons in vivo, including central(brain and spinal chord), peripheral (sympathetic, parasympathetic,sensory, and enteric neurons), and motorneurons. Accordingly, γ-HRG maybe utilized in methods for the diagnosis and/or treatment of a varietyof “neurologic diseases or disorders” which effect the nervous system ofa mammal, such as a human.

Such diseases or disorders may arise in a patient in whom the nervoussystem has been damaged by, e.g., trauma, surgery, stroke, ischemia,infection, metabolic disease, nutritional deficiency, malignancy, ortoxic agents. The agent is designed to promote the survival,proliferation or differentiation of neurons. For example, γ-HRG can beused to promote the survival or proliferation of motorneurons that aredamaged by trauma or surgery. Also, γ-HRG can be used to treatmotorneuron disorders, such as amyotrophic lateral sclerosis (LouGehrig's disease), Bell's palsy, and various conditions involving spinalmuscular atrophy, or paralysis. γ-HRG can be used to treat human“neurodegenerative disorders”, such as Alzheimer's disease. Parkinson'sdisease, epilepsy, multiple sclerosis, Huntington's chorea, Down'sSyndrome, nerve deafness, and Meniere's disease.

Further, γ-HRG can be used to treat neuropathy, and especiallyperipheral neuropathy. “Peripheral neuropathy” refers to a disorderaffecting the peripheral nervous system, most often manifested as one ora combination of motor, sensory, sensorimotor, or autonomic neuraldysfunction. The wide variety of morphologies exhibited by peripheralneuropathies can each be attributed uniquely to an equally wide numberof causes. For example, peripheral neuropathies can be geneticallyacquired, can result from a systemic disease, or can be induced by atoxic agent. Examples include but are not limited to distal sensorimotorneuropathy, or autonomic neuropathies such as reduced motility of thegastrointestinal tract or atony of the urinary bladder. Examples ofneuropathies associated with systemic disease include post-poliosyndrome; examples of hereditary neuropathies includeCharcot-Marie-Tooth disease, Refsum's disease, Abetalipoproteinemia,Tangier disease, Krabbe's disease, Metachromatic leukodystrophy, Fabry'sdisease, and Dejerine-Sottas syndrome; and examples of neuropathiescaused by a toxic agent include those caused by treatment with achemotherapeutic agent.

γ-HRG may also be used to treat muscle cells and medical conditionsaffecting them. For example, the γ-HRG may be used to treat apathophysiological condition of the musculature in a mammal, such as askeletal muscle disease (e.g. myopathy or dystrophy), a cardiac muscledisorder (such as atrial cardiac arrhythmias, cardiomyopathy, ischemicdamage, congenital disease, or cardiac trauma), or a smooth muscledisorder (for example, arterial sclerosis, vascular lesion, orcongenital vascular disease); to treat muscle damage; to decreaseatrophy of muscle cells; to increase muscle cell survival, proliferationand/or regeneration in a mammal; to treat hypertension; and/or to treata muscle cell which has insufficient functional acetylcholine receptors(as in a patient with myasthenia gravis or tachycardia).

γ-HRG may be used to induce the formation of ion channels in a surfacemembrane of a cell and/or for enhancing the formation of synapticjunctions in an individual. γ-HRG may be also useful as a memoryenhancer and may eliminate the “craving” for nicotine.

γ-HRG may be used to enhance repair and/or regeneration of tissues thatproduce ErbB receptor(s), especially the ErbB2 receptor. For example,γ-HRG may be used to treat dermal wounds; gastrointestinal disease;Barrett's esophagus; cystic or non-cystic end stage kidney disease; andinflammatory bowel disease. Similarly, this molecule may be used topromote reepithelialization in the human gastrointestinal, respiratory,reproductive or urinary tract.

In other embodiments, γ-HRG may be used to inhibit tumor cell invasionand metastasis. Tumors characterized by reduced endogenous γ-HRG levels(Park et al. Proc. Am. Assoc. Cancer Res. 34:521 (1993)) may beresponsive to γ-HRG. γ-HRG may be used to enhance chemotherapy byinteracting with ErbB receptors and thereby enhancing tumor cellsensitivity to a chemotherapeutic agent. It may be desirable to treatcarcinomas characterized by ErbB receptor overexpression using γ-HRG todirect a cytotoxic agent to the cancerous tissue. Examples of “cytotoxicagents” have been described above. γ-HRG-enzyme conjugates may bebeneficial for targeted prodrug therapy for targeting cells expressingErbB receptor(s).

In other situations, it may be desirable to treat the mammal with aγ-HRG antagonist, particularly where excessive levels of γ-HRG arepresent and/or excessive activation of ErbB receptors by γ-HRG isoccurring in the mammal. Exemplary conditions or disorders to be treatedwith a γ-HRG antagonist include benign or malignant tumors (e.g. renal,liver, kidney, bladder, breast, gastric, ovarian, colorectal, prostate,pancreatic, lung, vulval, thyroid, hepatic carcinomas; sarcomas;glioblastomas; and various head and neck tumors); leukemias and lymphoidmalignancies; other disorders such as neuronal, glial, astrocytal,hypothalamic and other glandular, macrophagal, epithelial, stromal andblastocoelic disorders; inflammatory, angiogenic and immunologicdisorders; psoriasis and scar tissue formation. γ-HRG antagonists mayalso be used to reverse resistance of tumor cells to theimmune-response, to inhibit pathological angiogenesis and to stimulatethe immune system.

In still further embodiments of the invention, γ-HRG antagonists may beadministered to patients suffering from neurologic diseases or disorderscharacterized by excessive production of γ-HRG and/or excessive ErbBreceptor activation by γ-HRG. γ-HRG antagonist may be used in theprevention of aberrant regeneration of sensory neurons such as may occurpost-operatively, or in the selective ablation of sensory neurons, forexample, in the treatment of chronic pain syndromes.

For therapeutic uses, the γ-HRG protein may be administered to a patientin need thereof. Alternatively, gene therapy (either nucleic acidencoding γ-HRG or, where it is desired to inhibit γ-HRG, antisensepolynucleotides) is contemplated herein.

Antisense inhibition of γ-HRG gene expression can occur at multiplelevels. The preferred approach however, is one which involvesinterfering with translation of γ-HRG mRNA into protein. To achievethis, one may use an oligodeoxynucleotide (oligo) complementary to theγ-HRG mRNA. This antisense oligo binds by complementary Watson-Crickbase pairing to the native sense mRNA. However, plasmid-derivedantisense RNA (i.e. wherein the antisense DNA is provided in a plasmid)is also contemplated. Mercola and Cohen Cancer Gene Therapy 2(1):47-59(1995). According to another embodiment, the so-called “anti-geneapproach”, the antisense molecule is one which binds to form a triplehelix or triplex with γ-HRG DNA via Hoogsteen (or anti-Hoogsteen)hydrogen bonding of the third base in the antisense molecule with thealready formed pair. Mercola and Cohen, supra. While antisense oligoscan be directed anywhere along the γ-HRG mRNA transcript, the preferredtarget sequence is at the 5′ end thereof, spanning the initiation codon.Generally, the antisense oligo will be relatively specific for γ-HRG andtherefore is complementary to at least a portion of the DNA encoding theunique NTD of γ-HRG. Oligos of interest will normally comprise at least15-17 bases. To identify the most active antisense sequence, deletionanalysis may be performed.

Antisense oligos can be readily synthesized by automated methods. SeeU.S. Pat. No. 5,489,677 issued Feb. 6, 1996. Normally the antisenseoligo intended for in vivo use will be modified so as to render it lesssusceptible to nuclease degradation and/or to improve the efficiencywith which it is taken up by a cell. Several backbone modifications havebeen developed to counteract nuclease degradation. One exemplarymodification results in a “methyl-phosphonate”(MO) oligo. According tothis approach, one of the nonbridging oxygen atoms in theinternucleotide bond is replaced with a methyl group which has the neteffect of eliminating the negative charge of the oligo. Tonkinson et al.Cancer Investigation 14(1):54-65 (1996). Another modification describedin Tonkinson et al. for reducing nuclease degradation is thephosphorothioate (PS) modification which involves replacing one of thenonbridging oxygens at the phosphorus with a sulfur. Other ways ofmodifying the basic oligo are reviewed in Cohen, J. Adv. Pharmacol.25:319-339 (1993). For example, a new analog has been reported whereinthe entire deoxyribose-phosphate backbone was replaced with apeptide-like backbone. See Mercola and Cohen, supra. To improve cellularuptake, the antisense oligos can be encapsulated in liposomes, complexedwith cationic lipids such as DOTMA, coupled to polylysine or lipofectin,and/or covalently attached to a cholesteryl moiety. See Tonkinson etal., supra.

There are two major approaches to getting the nucleic acid (optionallycontained in a vector) into the patient's cells; in vivo and ex vivo.For in vivo delivery the nucleic acid is injected directly into thepatient, usually at the site where the γ-HRG is required. For ex vivotreatment, the patient's cells are removed, the nucleic acid isintroduced into these isolated cells and the modified cells areadministered to the patient either directly or, for example,encapsulated within porous membranes which are implanted into thepatient (see, e.g. U.S. Pat. Nos. 4,892,538 and 5,283,187).

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. A commonly used vector for ex vivodelivery of the gene is a retrovirus.

The currently preferred in vivo nucleic acid transfer techniques includetransfection with viral vectors (such as adenovirus, Herpes simplex Ivirus, or adeno-associated virus) and lipid-based systems (useful lipidsfor lipid-mediated transfer of the gene are DOTMA, DOPE and DC-Chol, forexample). In some situations it is desirable to provide the nucleic acidsource with an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, and proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262:4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA 87:3410-3414 (1990). For review of the currently knowngene marking and gene therapy protocols see Anderson et al., Science256:808-813 (1992). See also WO 93/25673 and the references citedtherein.

Therapeutic formulations of γ-HRG or γ-HRG antagonist are prepared forstorage by mixing γ-HRG or γ-HRG antagonist having the desired degree ofpurity with optional physiologically acceptable carriers, excipients, orstabilizers (Remington's Pharmaceutical Sciences, 16th Edition, Osol.,A., Ed., (1980)), in the form of lyophilized cake or aqueous solutions.Pharmaceutically acceptable carriers, excipients, or stabilizers arenon-toxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween™, Pluronics™, or polyethylene glycol (PEG).

γ-HRG or γ-HRG antagonist to be used for in vivo administration must besterile. This is readily accomplished by filtration through sterilefiltration membranes, prior to or following lyophilization andreconstitution. The formulation ordinarily will be stored in lyophilizedform or in solution.

Therapeutic γ-HRG or γ-HRG antagonist compositions generally are placedinto a container having a sterile access port, for example, anintravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

The route of γ-HRG or γ-HRG antagonist administration is in accord withknown methods, e.g., injection or infusion by intravenous,intraperitoneal, intracerebral, intramuscular, intraocular,intraarterial, or intralesional routes, or by sustained-release systemsas noted below. γ-HRG is administered continuously by infusion or bybolus injection.

Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theprotein, which matrices are in the form of shaped articles, e.g., films,or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al., J. Biomed. Mater. Res., 15:167-277 (1981)and Langer, Chem. Tech., 12:98-105 (1982) or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers,22:547-556 (1983)), non-degradable ethylene-vinyl acetate (Langer etal., supra), degradable lactic acid-glycolic acid copolymers such as theLupron Depot™ (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyricacid (EP 133,988).

Sustained-release γ-HRG or γ-HRG antagonist compositions also includeliposomally entrapped drug. Liposomes containing γ-HRG are prepared bymethods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad.Sci. USA 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP142,641; Japanese patent application 83-118008; U.S. Pat. Nos. 4,485,045and 4,544,545; and EP 102,324. Ordinarily the liposomes are of the small(about 200-800 Angstroms) unilamellar type in which the lipid content isgreater than about 30 mol. % cholesterol, the selected proportion beingadjusted for the optimal therapy. Particularly useful liposomes can begenerated by the reverse phase evaporation method with a lipidcomposition comprising phosphatidylcholine, cholesterol andPEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes areextruded through filters of defined pore size to yield liposomes withthe desired diameter. A chemotherapeutic agent (such as Doxorubicin) isoptionally contained within the liposome. See Gabizon et al. J. NationalCancer Inst. 81(19)1484 (1989).

For neurologic diseases or disorders, it may be desirable to adsorbγ-HRG onto a membrane, such as a silastic membrane, which can beimplanted in proximity to damaged neural tissue, PCT Pub. No. WO91/04014 (published Apr. 4, 1991).

Other therapeutic regimens may be combined with the administration ofthe γ-HRG or γ-HRG antagonist of the instant invention. For thetreatment of neurological conditions, γ-HRG optionally is combined withor administered in concert with other neurotrophic factors to achieve adesired therapeutic effect. For example, γ-HRG may be used together withnerve growth factor (NGF), neurotrophins (NT-3), bone derived nervefactor (BDNF), neurotrophins-4 and -5 (NT-4/5), an insulin-like growthfactor (e.g., IGF-1 or IGF-2), gas6, or another neurotrophic factor toachieve a synergistic stimulatory effect on neurons, wherein the term“synergistic” means that the effect of the combination of γ-HRG with asecond substance is greater than that achieved with either substanceused individually. Suitable dosages for the neurotrophic factors may besimilar to those known in the art for such molecules.

The cancer patient to be treated with the γ-HRG or γ-HRG antagonistdisclosed herein may also receive radiation therapy. Alternatively, orin addition, a chemotherapeutic agent may be administered to thepatient. Preparation and dosing schedules for such chemotherapeuticagents may be used according to manufacturers' instructions or asdetermined empirically by the skilled practitioner. Preparation anddosing schedules for such chemotherapy are also described inChemotherapy Service Ed., M. C. Perry, Williams & Wilkins, Baltimore,Md. (1992). The chemotherapeutic agent may precede, or followadministration of the γ-HRG or γ-HRG antagonist or may be givensimultaneously therewith. For cancer indications, It may be desirable toalso administer antibodies against tumor associated antigens, such asantibodies which bind to EGFR, ErbB2, ErbB3, ErbB4, or vascularendothelial factor (VEGF). Alternatively, or in addition, one or morecytokines may be co-administered to the patient.

An effective amount of γ-HRG or γ-HRG antagonist to be employedtherapeutically will depend, for example, upon the therapeuticobjectives, the route of administration, and the condition of thepatient. Accordingly, it will be necessary for the therapist to titerthe dosage and modify the route of administration as required to obtainthe optimal therapeutic effect. A typical daily dosage might range fromabout 1 μg/kg to up to 100 mg/kg of patient body weight or more per day,preferably about 10 μg/kg/day to 10 mg/kg/day. Typically, the clinicianwill administer γ-HRG or γ-HRG antagonist until a dosage is reached thatachieves the desired effect for treatment of the above mentioneddisorders.

3. Non-Therapeutic Methods

γ-HRG polypeptide can be used for growing cells (such as glial andmuscle cells) ex vivo. It is desirable to have such populations of cellsin cell culture for isolation of cell-specific factors e.g. P75^(NGFR)which is a Schwann cell specific marker. Such factors are useful asdiagnostic tools or, in the case of P75^(NGFR), can be used an antigensto generate antibodies for diagnostic use. It is also beneficial to havepopulations of mammalian cells (e.g. Schwann cells) for use as cellularprostheses for transplantation into mammalian patients (e.g. into areasof damaged spinal cord in an effort to influence regeneration ofinterrupted central axons, for assisting in the repair of peripheralnerve injuries and as alternatives to multiple autografts).

In accordance with the in vitro methods of the invention, cellscomprising an ErbB receptor are provided and placed in a cell culturemedium. Suitable tissue culture media are well known to persons skilledin the art and include, but are not limited to, Minimal Essential Medium(MEM), RPMI-1640, and Dulbecco's Modified Eagle's Medium (DMEM). Thesetissue culture medias are commercially available from Sigma ChemicalCompany (St. Louis, Mo.) and GIBCO (Grand Island, N.Y.). The cells arethen cultured in the cell culture medium under conditions sufficient forthe cells to remain viable and grow in the presence of an effectiveamount of γ-HRG. The cells can be cultured in a variety of ways,including culturing in a clot, agar, or liquid culture.

The cells are cultured at a physiologically acceptable temperature suchas 37° C., for example, in the presence of an effective amount of γ-HRG.The amount of γ-HRG may vary, but preferably is in the range of about 10ng/ml to about 1 mg/ml. The γ-HRG can of course be added to the cultureat a dose determined empirically by those in the art without undueexperimentation. The concentration of γ-HRG in the culture will dependon various factors, such as the conditions under which the cells andγ-HRG are cultured. The specific temperature and duration of incubation,as well as other culture conditions, can be varied depending on suchfactors as, e.g., the concentration of the γ-HRG, and the type of cellsand medium. Those skilled in the art will be able to determine operativeand optimal culture conditions without undue experimentation.

Techniques for culturing Schwann cells ex vivo are described in Li etal., supra and Sklar et al., supra describe ex vivo culturing of clonalhuman myoblasts. γ-HRG can replace the other heregulin polypeptides usedin these methods.

γ-HRG polypeptide can be used in the diagnosis of cancers characterizedby erbB (e.g. erbB2) overexpression and/or amplification. Similarly,molecules which detect γ-HRG expression (e.g. nucleic acid probes andanti-γ-HRG antibodies) can be used to detect γ-HRG expression (e.g. incancer where γ-HRG leads to the formation of a constitutive activecomplex, such as some breast cancers, see Example below). Suchdiagnostic assay(s) can be used in combination with otherdiagnostic/prognostic evaluations such as determining lymph node status,primary tumor size, histologic grade, estrogen or progesterone status,tumor DNA content (ploidy), or cell proliferation (S-phrase fraction).See Muss et al., New Eng. J. Med., 330(18):1260-1266 (1994).

The sample as herein defined is obtained, e.g. tissue sample from theprimary lesion of a patient. Formalin-fixed, paraffin-embedded blocksare prepared. See Muss et al., supra and Press et al., Cancer Research54:2771-2777 (1994). Tissue sections (e.g. 4 μM) are prepared accordingto known techniques. The extent of γ-HRG binding to the tissue sectionsis then quantified.

Generally, the γ-HRG (protein or nucleic acid probe) or HRG antibodywill be labelled either directly or indirectly with a detectable label.Numerous labels are available which can be generally grouped into thefollowing categories:

(a) Radioisotopes, such as ³⁵S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹ I. The γ-HRG orantibody can be labeled with the radioisotope using the techniquesdescribed in Current Protocols in Immunology, Ed. Coligen et al., WileyPublishers, Vols 1 & 2, for example, and radioactivity can be measuredusing scintillation counting.

(b) Fluorescent labels such as rare earth chelates (europium chelates)or fluorescein and its derivatives, rhodamine and its derivatives,dansyl, Lissamine, phycoerythrin and Texas Red are available. Thefluorescent labels can be conjugated to the γ-HRG or antibody using thetechniques disclosed in Current Protocols in Immunology, supra, forexample. Fluorescence can be quantified using a fluorimeter (Dynatech).

(c) Various enzyme-substrate labels are available and U.S. Pat. No.4,275,149 provides a review of some of these. The enzyme generallycatalyses a chemical alteration of the chromogenic substrate which canbe measured using various techniques. For example, the enzyme maycatalyze a color change in a substrate, which can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Techniques forquantifying a change in fluorescence are described above. Thechemiluminescent substrate becomes electronically excited by a chemicalreaction and may then emit light which can be measured (using a DynatechML3000 chemiluminometer, for example) or donates energy to a fluorescentacceptor. Examples of enzymatic labels include luciferases (e.g.,firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase,β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to proteins are described in O'Sullivan et al.,Methods for the Preparation of Enzyme-Antibody Conjugates for use inEnzyme Immunoassay, in Methods in Enzym. (ed J. Langone & H. VanVunakis), Academic press, New York, 73:147-166 (1981) and CurrentProtocols in Immunology, supra.

Examples of enzyme-substrate combinations include, for example: (a)Horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate,wherein the hydrogen peroxidase oxidizes a dye precursor (e.g.orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidinehydrochloride (TMB)); (b) alkaline phosphatase (AP) withpara-Nitrophenyl phosphate as chromogenic substrate; and (c)β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g.p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate4-methylumbelliferyl-β-D-galactosidase.

Numerous other enzyme-substrate combinations are available to thoseskilled in the art. For a general review of these, see U.S. Pat. Nos.4,275,149 and 4,318,980.

Sometimes, the label is indirectly conjugated with the γ-HRG orantibody. The skilled artisan will be aware of various techniques forachieving this. For example, the γ-HRG or antibody can be conjugatedwith biotin and any of the three broad categories of labels mentionedabove can be conjugated with avidin, or vice versa. Biotin bindsselectively to avidin and thus, the label can be conjugated with theγ-HRG or antibody in this indirect manner. See, Current Protocols inImmunology, supra, for a review of techniques involving biotin-avidinconjugation. Alternatively, to achieve indirect conjugation of the labelwith the γ-HRG or antibody, the γ-HRG or antibody is conjugated with asmall hapten (e.g. digoxin) and one of the different types of labelsmentioned above is conjugated with an anti-hapten antibody (e.g.anti-digoxin antibody). Thus, indirect conjugation of the label with theγ-HRG or antibody can be achieved.

In another embodiment of the invention, the γ-HRG or antibody need notbe labeled, and the presence thereof can be detected using a labeledanti-γ-HRG or anti-antibody antibody (e.g. conjugated with HRPO).

In the preferred embodiment, the γ-HRG or antibody is labeled with anenzymatic label which catalyzes a color change of a substrate (such astetramethyl benzimidine (TMB), or orthaphenylene diamine (OPD)). Thus,the use of radioactive materials is avoided. A color change of thereagent can be determined spectrophotometrically at a suitablewavelength (e.g. 450 nm for TMB and 490 nm for OPD, with a referencewavelength of 650 nm).

Thus, the tissue sections on slides are exposed to the labelled γ-HRG orantibody and the intensity of staining of the tissue sections isdetermined. While in vitro analysis is normally contemplated, in vivodiagnosis using γ-HRG or antibody conjugated to a detectable moiety(e.g. In for imaging) can also be performed. See, e.g., U.S. Pat. No.4,938,948.

γ-HRG preparations are also useful as standards in assays for γ-HRG(e.g., by labeling γ-HRG for use as a standard in a radioimmunoassay,enzyme-linked immunoassay, or radioreceptor assay), in affinitypurification techniques (e.g. for an ErbB receptor such as ErbB3 orErbB4 receptor), and in competitive-type receptor binding assays whenlabeled with radioiodine, enzymes, fluorophores, spin labels, and thelike. γ-HRG polypeptides are also useful as immunogens for generatinganti-γ-HRG antibodies for diagnostic use. In this respect the unique NTD(or fragments thereof, (e.g., having a consecutive sequence of 20 ormore amino acid residues) are useful as immunogens for generatingγ-HRG-specific antibodies for use in its detection and/or purification.

Similarly, the nucleic acid encoding γ-HRG is useful as a probe fordetecting γ-HRG expression in various tissues. In this respect, nucleicacid encoding or complementary to nucleic acid encoding the unique NTDof γ-HRG or a fragment thereof is particularly useful. Techniques forDNA analysis are well known. See, e.g. U.S. Pat. No. 4,968,603.Normally, the DNA analysis will involve Southern blotting a samplederived from a mammal.

4. γ-HRG Antibodies & Uses Thereof

Techniques for generating antibodies, such as polyclonal and monoclonalantibodies are well known in the art. Polyclonal antibodies generallyare raised by immunizing animals with γ-HRG or a fragment thereof(optionally conjugated to a heterologous protein that is immunogenic inthe species to be immunized). Monoclonal antibodies directed towardγ-HRG may be produced using any method which provides for the productionof antibody molecules by continuous cell lines in culture. Examples ofsuitable methods for preparing monoclonal antibodies include theoriginal hybridoma method of Kohler et al., Nature 256:495-497 (1975),and the human B-cell hybridoma method, Kozbor, J., Immunol. 133:3001(1984); Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); andBoerner et al., J. Immunol. 147:86-95 (1991).

DNA encoding the monoclonal antibodies of the invention is readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of antibodies). The hybridoma cellsof the invention serve as a preferred source of such DNA. Once isolated,the DNA may be placed into expression vectors, which are thentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, to obtain the synthesis ofmonoclonal antibodies in the recombinant host cells.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofthe homologous murine sequences (Morrison, et al., Proc. Natl. Acad.Sci. USA 81:6851 (1984)), or by covalently joining to the immunoglobulincoding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. In that manner, “chimeric” or “hybrid”antibodies are prepared that have the binding specificity of ananti-γ-HRG monoclonal antibody herein.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); and Verhoeyen et al., Science 239:1534-1536 (1988)),by substituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody (Sims et al., J.Immunol, 151:2296 (1993); and Chothia and Lesk, J. Mol. Biol. 196:901(1987)). Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA 89:4285 (1992); and Presta et al., J. Immunol. 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

According to an alternative method for producing human antibodies,transgenic animals (e.g., mice) are available that are capable, uponimmunization, of producing a full repertoire of human antibodies in theabsence of endogenous immunoglobulin production. For example, it hasbeen described that the homozygous deletion of the antibody heavy-chainjoining region (J_(H)) gene in chimeric and germ-line mutant miceresults in complete inhibition of endogenous antibody production.Transfer of the human germ-line immunoglobulin gene array in suchgerm-line mutani mice will result in the production of human antibodiesupon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad.Sci. USA, 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993);and Bruggermann et al., Year in Immuno. 7:33 (1993).

Alternatively, phage display technology (McCafferty et al., Nature348:552-553 (1990)) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats; for their review see, e.g., Johnson, Kevin S. andChiswell, David J., Current Opinion in Structural Biology 3:564-571(1993). Several sources of V-gene segments can be used for phagedisplay. Clackson et al., Nature, 352:624-628 (1991) isolated a diversearray of anti-oxazolone antibodies from a small random combinatoriallibrary of V genes derived from the spleens of immunized mice. Arepertoire of V genes from unimmunized human donors can be constructedand antibodies to a diverse array of antigens (including self-antigens)can be isolated essentially following the techniques described by Markset al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al., EMBO J.12:725-734 (1993).

Bispecific antibodies are antibodies that have binding specificities forat least two different antigens. In the present case, one of the bindingspecificities is for a γ-HRG, the other one is for any other antigen,e.g., for another polypeptide that activates an ErbB receptor. Forexample, bispecific antibodies specifically binding γ-HRG and anotherheregulin polypeptide are within the scope of the present invention.Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g. F(ab′)₂ bispecific antibodies).

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is preferred to havethe first heavy-chain constant region (CH1) containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach, the interface between a pair of antibodymolecules can be engineered to maximize the percentage of heterodimerswhich are recovered from recombinant cell culture. The preferredinterface comprises at least a part of the C_(H)3 domain of an antibodyconstant domain. In this method, one or more small amino acid sidechains from the interface of the first antibody molecule are replacedwith larger side chains (e.g. tyrosine or tryptophan). Compensatory“cavities” of identical or similar size to the large side chain(s) arecreated on the interface of the second antibody molecule by replacinglarge amino acid side chains with smaller ones (e.g. alanine orthreonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360). Heteroconjugate antibodies may be made using any convenientcross-linking methods. Suitable cross-linking agents are well known inthe art, and are disclosed in U.S. Pat. No. 4,676,980, along with anumber of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science 229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent sodiumarsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175:217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)Fnolecule.Each Fab′ fragment was separately secreted from E. coli and subjected todirected chemical coupling in vitro to form the bispecific antibody. Thebispecific antibody thus formed was able to bind to cells overexpressingerbB and normal human T cells, as well as trigger the lytic activity ofhuman cytotoxic lymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al. Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al. J. Immunol. 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147:60(1991).

To manufacture a neutralizing antibody, antibodies are made using thetechniques for generating these molecules elaborated above. Thepreferred neutralizing antibody is specific for γ-HRG (i.e. does notsignificantly cross-react with other heregulins as determined byimmuno-precipitation, for example). Following production of a panel ofantibodies, the antibodies are subjected to a screening process in orderto identify those molecules which meet the desired criteria (i.e. whichare able to neutralize a biological activity of γ-HRG either in vitro orin vivo). For example, the ability of the γ-HRG to block γ-HRG activityin any one or more of the assays described above for screening for γ-HRGvariants can be evaluated. Those antibodies which block the ability ofγ-HRG to bind to and/or activate an ErbB receptor and/or the mitogenicactivity of γ-HRG on cells can be selected as neutralizing antibodies.

The antibodies may be coupled to a cytotoxic agent or enzyme (e.g. aprodrug-activating enzyme) in a similar manner to that described abovefor γ-HRG polypeptide. Furthermore, the antibodies may be labelled asdescribed above for γ-HRG polypeptides, especially where the antibodiesare to be used in diagnostic assays.

γ-HRG antibodies are useful in diagnostic assays for γ-HRG, e.g., itsproduction in specific cells, tissues, or serum. The antibodies arelabeled in the same fashion as γ-HRG described above and/or areimmobilized on an insoluble matrix. Suitable diagnostic assays for γ-HRGantibodies are well known an have been discussed above with respect toγ-HRG polypeptide assays. Given the secreted nature of the γ-HRGmolecule, a particularly useful assay for detecting this molecule inbiological fluids is as described in U.S. Pat. No. 5,401,638, issuedMar. 28, 1995. According to this approach, the γ-HRG in the bodily fluid(e.g. human plasma or serum) is “captured” using an immobilized antibodywhich binds specifically thereto. A second antibody, which is optionallylabelled, is then used to detect the captured γ-HRG. This secondantibody may be one which binds any epitope of the γ-HRG. Thus, theamount of γ-HRG in the bodily fluid relative to a control can bedetected.

γ-HRG antibodies are also useful for affinity purification of γ-HRG fromrecombinant cell culture or natural sources.

Neutralizing anti-γ-HRG antibodies may also be used to block γ-HRGbiological activity in vitro or in vivo. Clinical situations in whichthis may be desirable are discussed above with respect to uses for γ-HRGantagonists.

5. Diagnostic Kits & Articles of Manufacture

Since the invention provides at least two types of diagnostic assay(i.e. for detecting cancer using γ-HRG and for detecting the presence ofγ-HRG in a sample using antibodies or DNA markers) as a matter ofconvenience, the reagents for these assays can be provided in a kit,i.e., a packaged combination of reagents, for combination with thesample to be tested. The components of the kit will normally be providedin predetermined ratios. Thus, a kit may comprise the antibody or γ-HRG(DNA or polypeptide) labelled directly or indirectly with a suitablelabel. Where the detectable label is an enzyme, the kit will includesubstrates and cofactors required by the enzyme (e.g. a substrateprecursor which provides the detectable chromophore or fluorophore). Inaddition, other additives may be included such as stabilizers, buffersand the like. The relative amounts of the various reagents may be variedwidely to provide for concentrations in solution of the reagents whichsubstantially optimize the sensitivity of the assay. Particularly, thereagents may be provided as dry powders, usually lyophilized, includingexcipients which on dissolution will provide a reagent solution havingthe appropriate concentration. The kit also suitably includesinstructions for carrying out the bioassay.

In another embodiment of the invention, an article of manufacturecontaining materials useful for the treatment of the disorders describedabove is provided. The article of manufacture comprises a container anda label. Suitable containers include, for example, bottles, vials,syringes, and test tubes. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is effective for treating the condition and may have a sterileaccess port (for example the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle). The active agent in the composition is γ-HRG or an antagonistthereof. The label on, or associated with, the container indicates thatthe composition is used for treating the condition of choice. Thearticle of manufacture may further comprise a second containercomprising a pharmaceutically-acceptable buffer, such asphosphate-buffered saline, Ringer's solution and dextrose solution. Itmay further include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters. needles,syringes, and package inserts with instructions for use.

The following examples are offered by way of illustration and not by wayof limitation. The disclosures of all citations in the specification areexpressly incorporated herein by reference.

Example 1

This example describes the isolation and biochemical characterization ofthe γ-HRG polypeptide of the present invention.

Materials & Methods

Reagents: The EGF-like domain of HRGβ1₍₁₇₇₋₂₄₄₎ was expressed in E.coli, purified and radioiodinated as described previously (Sliwkowski etal. J. Biol. Chem. 269:14661-14665 (1994)). The anti-ErbB2 monoclonalantibodies 2C4 and 4D5 have been described elsewhere (Fendly et al.Cancer Research 50:1550-1558 (1990)).

ErbB3- and ErbB4-immunoadhesins: A unique MI I site was engineered intoa plasmid expressing human IgG heavy chain at the region encoding thehinge domain of the immunoglobulin. MI I sites were also engineered intoa set of ErbB expression plasmids at the region encoding the ECD/TMjunctions of these receptors. All mutageneses were done using the Kunkelmethod (Kunkel, T., Proc. Natl. Acad. Sci. U.S.A. 82:488 (1985)). The MII sites were utilized to make the appropriate ErbB-IgG fusionconstructs. The fusion junctions of the various ErbB-IgG chimeras were:for ErbB2, E⁶⁴⁶ _(ErbB2)-(TR)-DKTH²²⁴ _(VH); for ErbB3, L⁶³⁶_(ErbB3)-(TR)-DKTH²²⁴ _(VH); for ErbB4, G⁶⁴⁰ _(ErbB4)-(TR)-DKTH²²⁴_(VH). The conserved TR sequence is derived from the MI I site. Thefinal expression constructs were in a pRK-type plasmid backbone whereineukaryotic expression is driven by a CMV promoter (Gorman et al., DNAProt. Eng. Tech. 2:3-10 (1990)).

To obtain protein for in vitro experiments, adherent HEK-293 cells weretransfected with the appropriate expression plasmids using standardcalcium phosphate methods (Gormar et al., supra and Huang et al.,Nucleic Acids Res. 18:937-947 (1990)). Serum-containing media wasreplaced with serum-free media 15 hours post-transfection and thetransfected cells incubated for 5-7 days. The resulting conditionedmedia was harvested and passed through Protein A columns (1 mL PharmaciaHiTrap™). Purified IgG fusions were eluted with 0.1 M citric acid (pH4.2) into tubes containing 1 M Tris pH 9.0. The eluted proteins weresubsequently dialyzed against PBS and concentrated using Centri-prep-30filters (Amicon). Glycerol was added to a final concentration of 25% andthe material stored at −20° C. Concentrations of material weredetermined via a Fc-ELISA.

Cell Culture: Human breast cancer cell lines MDA-MB-175, MDA-MB-231,SK-BR-3 and MCF7 were obtained from the American Type Culture Collectionand maintained in a 50:50 mixture of F12 Ham's and Dulbecco's modifiedEagle medium (DMEM), supplemented with 10% heat inactivated FBS, 2 mMglutamine and 10% penicillin-streptomycin.

Generation and Characterization of cDNA Library: Total RNA was purifiedfrom MDA-MB-175 cells using the guanidinium isothiocyanate-cesiumchloride procedure (Sambrook et al., Molecular Cloning: A LaboratoryManual (New York: Cold Spring Harbor Laboratory Press, (1989)). Poly(A)⁺ RNA was isolated using oligo (dT) Dynabeads (DYNAL) as recommendedby the supplier. First and second strand syntheses were performed usinga Gibco BRL cDNA synthesis kit. λgt10 cDNA recombinants were generatedwhen a cDNA cloning system from Amersham was used. In vitro packagingwas performed using Gigapack II™ packaging extract (Stratagene).Pstl-Xhol HRGβ3 cDNA fragment (nt 144-618) was labeled by random primingand 1×10⁶ plaques were screened. Positive clones were confirmed andpurified by secondary and tertiary screening. Phage DNA was isolated asa BamHI fragment and subcloned into the corresponding site ofpBluescript SK−. Clone 5 was completely sequenced using the Sequenaseversion 2.0™ DNA sequencing kit (United States Biochemicals, Inc.). Bothstrands were sequenced.

Bacterial Expression System: A cDNA fragment of clone 5 (nt 1690-2722)was subcloned into the pET-32 TRX fusion vector (Novagen). ThisBgIII-BgIII fragment was inserted into the BamHI site of the pET32aplasmid. The trx γ-HRG (amino acids 455-768) protein expression in E.coli was induced as recommended by the supplier.

Purification of Recombinant γ-HRG: E. coli cells expressing trx γ-HRGwere collected and suspended at 9 ml/g in 50 mM Tris HCL pH 8. Lysozymewas added to a final concentration of 0.2 mg/ml and the solution wasstirred on ice for 1 hr. Dnase I (10 μg/ml) and MgCl₂ (4 mM) were added.The solution was then sonicated for 30 min and cell pellets collectedafterwards. The pellet fraction was dissolved at 250 ml/g in 6 M GdnHCL, 0.1 M Tris HCL, pH 8.8. Solubilized proteins were sulfitolyzed byadding 1/10 volume of 1 M Na₂SO₃ and 1/10 volume of 0.2 M Na₂S₄O₆. Thereaction was allowed to proceed for 1.5 hours at room temperature andprotein was purified by gel filtration chromatography using a High LoadSuperdex™ 75 prep grade column (Pharmacia). Refolding was initiated bythe addition of 1 mM cysteine, and 10 mM methionine was added as anantioxidant and incubated overnight at room temperature. Proteinconcentration was determined by quantitative amino acid analysis.

Northern and Southern Hybridization: Total RNA was isolated by themethod of Chomczynski et al. Anal Biochem. 162:156-159 (1987). Poly (A)⁺was isolated using oligo d(T) cellulose columns (Qiagen) as recommendedby the supplier. RNA was denatured and size fractioned in a 0.8%formaldehyde/1% agarose gel and transferred onto nylon membrane (Hybond,Amersham). RNA was UV crosslinked (UV Stratalinker, Stratagene).Prehybridization was carried out at 42° C. in 50% formamide/1% SDS/1 MNaCl, 10% dextransulfate and 100 μg/ml herring sperm DNA for at least 2hours. cDNA probes using either a restriction fragment withcomplementary sequence to the EGF-like domain of HRGβ3 or a Kpnl-AvallcDNA fragment encoding the unique sequence of γ-HRG (nt 1238-1868) wereradiolabeled by random priming (Prime-It II, Stratagene). Hybridizationwas done in equal solution at 42° C. containing the ³²P labeledfragments for 16 hr. Blots were washed several times with 2×SSC/1% SDSat room temperature, washed with the same solution at 65° C. for 20 minand finally washed with 0.2×SSC/0.1% SDS at room temperature for 15 min.The blots were air dried and exposed to Du Pont Reflection™ film withintensifying screens at −80° C. for 7-40 hours. Human multiple tissueNorthern blots (Clontech) containing 2 μg poly (A)⁺ from spleen, thymus,prostate, testis, ovary, small intestine, colon, peripheral bloodleukocytes, heart, brain, placenta, lung, liver, skeletal muscle, kidneyand pancreas were hybridized with a radiolabeled γ-HRG cDNA probe (nt841-1447) as recommended by the supplier.

MDA-MB-175 and MDA-MB-231 genomic DNA was isolated as described inSambrook et al, supra. DNA was digested with different restrictionenzymes, prior to transfer treated with 0.25 N HCl and transferred ontonylon membrane (Hybond, Amersham). BgIII-Ndel cDNA fragment of γ-HRG (nt1690-2351) was also radiolabeled by random priming and used as ahybridization probe. Prehybridization was carried out in6×SSC/5×Denhardt's/0.75% SDS, 10% Dextransulfate and 100 μg/ml herringsperm DNA at 68° C. for 4 hours and hybridization with radiolabeledprobe was done overnight. The same wash conditions as for Northern blotswere used except a wash step with 0.2×SSC/0.1% SDS at 68° C. was addedand detection was pursued as described above.

¹²⁵I-HRG Binding Assay: Binding assays were performed in Nunc breakapartstrip wells. Plates were coated at 4° C. overnight with 100 μl of 5μg/ml goat-anti-human antibody (Boehringer Mannheim) in 50 mM carbonatebuffer (pH 9.6). Plates were rinsed twice with wash buffer (PBS/0.05%Tween-20™) and blocked with 100 μl 1% BSA/PBS for 30 min. Buffer wasremoved and each well was incubated with 15 ng IgG fusion protein in 1%BSA/PBS under vigorous shaking for 1.5 hours. Plates were rinsed threetimes with wash buffer and competitive binding was carried out by addingvarious amounts of γ-HRG and ¹²⁵I-HRGβ1 under vigorous shaking. Afterincubation for 1.5-2 hours, wells were rinsed three times with washbuffer, drained and individual wells were counted using a 100 Series IsoData γ-counter.

Tyrosine Phosphorylation Assay: MCF7 cells were plated in 24 well platesat 1×10⁵ cells/well in F12/DMEM containing 10% FBS. After 48 hours,cells were washed with serum free F12/DMEM and serum starved for 6hours. Various concentrations of bacterial expressed truncated γ-HRG(i.e., 0 pM, 22 pM, 66 pM, 200 pM and 600 pM trx γ-HRG) or unpurifiedconditioned medium of MDA-MB-175 cells were prepared in binding buffer(0.1% BSA in F12/DMEM) and added to each well. After 8 min incubation atroom temperature, media was carefully aspirated and reactions werestopped by adding 100 μl of sample buffer (5% SDS, 0.25%2-mercaptoethanol, 25 mM Tris-HCL pH 6.8). 20 μl of each sample was sizefractionated in a 4-12% gradient gel (Novex) and thenelectrophoretically transferred onto nitrocellulose membrane.Antiphosphotyrosine (4G10, from UBI, used at 1 μg/ml) immunoblots weredeveloped and the predominant reactive band at M, ˜180 kDa wasquantified by reflectance densitometry.

Production and characterization of conditioned medium from MDA-MB-175cells. Cells were seeded in T175 flasks and grown until reaching 70-80%confluency (˜2.5×10⁷ cells/flask). Subsequently, cells were washed withPBS and grown in serum free F12/DMEM medium for 3-4 days. Medium wasthen collected, filtered and concentrated using an ultrafiltration cellwith YM10 Diaflo ultafiltration membranes (Amicon). γ-HRG was visualizedin conditioned medium of MDA-MB-175 cells by Western blot analysis undernon reducing conditions. γ-HRG was partially purified by HPLC using a C4reverse phase column. CHO expressed full length HRGβ1 (lane 1) and semipure γ-HRG (lane 2) were electrophoresed, blot was probed withErbB2/ErbB4 IgG heterodimers and Western blot was developed. A ˜64 kDaband could be seen in the lane containing partial purified supernatantwhereas CHO expressed full length HRGβ1 migrated as a 45 kDa protein.

Cell Proliferation Assay with Crystal Violet: Tumor cell lines wereplated in 96 well plates at following densities: 2×10⁴ cells/well forMDA-MB-175 and 1×10⁴ cells/well for SK-BR-3. The media contained 1% FBSand cells were allowed to adhere for 2 hours. Monoclonal antibodies,immunoadhesions (10 μg/ml) or media alone were added and the cells wereincubated for 2 hours at 37° C. rHRGβ1₁₇₇₋₂₄₄ was added at a finalconcentration of 1 nM, or 100 nM for neutralising the immunoadhesion,and the cells were incubated for 4 days. Monolayers were washed with PBSand stained/fixed with 0.5% crystal violet. Plates were air dried, thedye was eluted with 0.1 M sodium citrate (pH 4.2) in ethanol (50:50) andthe absorbance was measured at 540 nm.

Results & Discussion

Isolation and sequence analysis of γ-HRG: To characterize the heregulintranscript in MDA-MB-175 cells, a λgt 10 cDNA library was constructedwith mRNA derived from this cell line. The library was screened with acDNA probe corresponding to the EGF-like domain and part of theN-terminal sequence of HRGβ3. Various clones were identified. One of theclones which appeared to contain the full length cDNA sequence wasisolated and sequenced. FIG. 1 shows the nucleotide sequence and thepredicted amino acid sequence of γ-HRG. The single open reading frame of2303 bp starts with an ATG codon at nt 334. This start codon lies in anucleotide sequence context, which is known to be a potentialtranslation initiation site (Kozak, Nucleic Acid Research 15:8125-8148(1987)). Several termination codons were found upstream of theinitiation codon. The stop codon TAG at nt 2637 is followed by the 3′noncoding sequence, which is identical to other HRG isoform sequencesand includes a polyadenylation signal followed by an A-rich region. Theoper reading frame encodes a protein of 768 amino acid residues with acalculated molecular mass of 84.2 kDa.

Structural analysis of γ-HRG: γ-HRG as shown in FIG. 2 has an EGF-likedomain which is completely identical to GGF, SMDF and the HRGβ isoforms.Like GGF, SMDF and HRGβ3 the amino acid sequence ends in thejuxtamembrane region after a stretch of 11 variable amino acid residues.Therefore γ-HRG lacks the transmembrane region and the cytoplasmicdomain. As in HRGα and HRGβ, the spacer region with many potential N-and O-linked glycosylation sites is present. This region connects theEGF-like domain with the Ig-like domain. Except for the recentlydiscovered new isoform, named sensory and motor neuron derived factor(SMDF), all known HRG forms contain this motif. The N-terminal regionupstream of the Ig-like domain is unique and distinct from all otherheregulins. The first 33 amino acids which are found in HRGα and HRGβare absent. Instead γ-HRG possesses a 560 amino acid sequence, whichshows no similarity to any known DNA or protein sequences. As seen withother HRG isoforms, γ-HRG lacks an N-terminal signal sequence. However,the unique region shows a 22 amino acid stretch of hydrophobic aminoacid residues, which may function as an internal signal sequence and maybe responsible for the secretion ability of γ-HRG. No structural motifscould be found in the N-terminal region after searching a motifdatabase.

Identification of heregulin transcripts in MDA-MB-175, MDA-MB-231 celllines and different human tissues: To compare the heregulin transcriptsin MDA-MB-175, MDA-MB-231 cell lines and mammary gland tissue, aNorthern blot analysis was performed on poly(A)⁺ RNA. By using aradiolabeled fragment corresponding to the EGF-like domain of HRGβ3,three transcripts were detected in MDA-MB-231 RNA as observed before(Holmes et al., Science 256:1205-1210 (1992)). The variability in signalintensities of the three transcripts is due to different cell batchesand RNA preparations. The same transcript sizes were also seen in normalbreast tissue RNA. However in MDA-MB-175 RNA only one major transcriptof 3.3 kb could be found. When a ³²P labeled cDNA probe corresponding tothe unique N-terminal sequence of γ-HRG was used, no hybridizationsignals could be located either in MDA-MB-231 RNA nor in breast tissueRNA. In MDA-MB-175 RNA again the major 3.3 kb band could be detected.Due to the overexposure of the autoradiogram, bands of much lowerintensities with the sizes of 1.8 kb, 5 kb and 7.5 kb could be seen.They were also present in autoradiograms after extensive exposure usingthe EGF-like domain probe. Various human tissues were examined for thepresence of γ-HRG mRNA. Transcripts were found in testis, ovary,skeletal muscle and in lower intensity in heart, brain and kidney. Notranscripts could be found by Northern blot analysis in spleen, thymus,prostate, small intestine, colon, peripheral blood leukocytes, placenta,lung, liver and pancreas. The mRNA in the expressing tissues varied bysizes. Without being bound to any theory, this is probably due todifferential splicing events in these tissues.

The γ-HRG isoform is not a result of DNA rearrangement: Theheregulin/NDF isoforms are products of a differentially spliced gene.The possibility that γ-HRG may not be an alternative splice variant ofthe heregulin gene, but a product of DNA rearrangement in MDA-MB-175cells was addressed. To answer this question, Southern blot analysis ofgenomic DNA retrieved from MDA-MB-231 and MDA-MB-175 cells wasperformed. Genomic DNA of both cell lines was digested with restrictionenzymes and fractionated by size. The blot was hybridized with aradiolabeled cDNA probe (nt 1690-2351) of γ-HRG. The 5′ prime of thecDNA probe was complementary to the unique N-terminal sequence. The 3′prime contained a part of the commonly shared sequence of theheregulins, which Marchionni et al. supra defined as exon 2. This meansthat the probe was designed over the exon/intron junction or possibleDNA rearrangement site. Comparison of the band sizes in both MDA-MB-175and MDA-MB-231 cell lines revealed no differences, which is a strongindication that γ-HRG is not a product of DNA rearrangement, but rathera new splice variant of the heregulin gene.

Functional activity of γ-HRG. When medium conditioned by MDA-MB-175cells was analyzed in a binding assay on MCF7 cells a concentration of˜26 pM could be detected. However this number was obtained by comparingthe displacement characteristics of unpurified medium to HRGβ1₍₁₇₇₋₂₄₄₎binding. Due to this low concentration in the supernatant, therecombinant protein was produced. Partial sequence analysis of differentλgt 10 clones, obtained after screening with the EGF-like domain, led tothe conclusion that MDA-MB-175 cells transcribe more than one novel HRGisoform. Aside from γ-HRG of FIG. 1 (SEQ ID NO:2), clone 20 encoding anisoform of the FIG. 1 γ-HRG sequence was extensively characterized (seeFIGS. 5 and 6A-G; SEQ ID NOS. 10 and 11, respectively). Compared to theγ-HRG sequence, clone 20 contains at least one insert of 26 amino acidresidues between amino acid 560 and 561. Furthermore the N-terminal DNAsequence varies from γ-HRG by additional base pairs. However clone 20lacks the 5′ end. Initial expression experiments in mammalian cells weredone with a construct containing clone 20 DNA sequence. Protein sequenceanalysis of the secreted form showed a processed polypeptide, lackingthe N-terminal sequence and hydrophobic region. Proteolytic cleavageoccurred in the additional insert region of this clone between twoarginine residues (i.e. the N-terminal residue of the proteolyticallyprocessed protein was residue 315 in FIG. 5; SEQ ID NO:10). Based onthese data, a N-terminal truncated version of γ-HRG thioredoxin fusionprotein was expressed in an bacterial expression system. Although it hasbeen reported that several mammalian cytokines and growth factors staysoluble in the E. coli cytoplasm, when expressed as a C-terminalthioredoxin fusion protein, trx γ-HRG was insoluble (La Vallie et al.Bio/Technology 11: 187-193 (1993)). Under those conditions trx γ-HRGaccumulated in inclusion bodies from which the recombinant protein wasisolated. Following purification, the protein was sulfitolyzed and thenrefolded by the addition of cysteine. The interaction of γ-HRG with theheregulin receptors ErbB3 and ErbB4 was examined. Binding analysis wasperformed using an in vitro system, so that the binding characteristicsof the individual receptors could be studied. IgG fusion proteinscontaining the extracellular domain of ErbB3 or ErbB4 were constructed.The immunoadhesins were incubated with ¹²⁵I-HRGβ1₍₁₇₇₋₂₄₄₎ (0.23 nM) andincreasing amounts of unlabeled γ-HRG. γ-HRG was able to displace¹²⁵I-HRGβ1₍₁₇₇₋₂₄₄₎ binding to both receptors respectively ErbB3 andErbB4. Binding analysis revealed an EC₅₀ of 19±1.3 nM for γ-HRG to ErbB3immunoadhesins and an EC₅₀ of 13.3±0.8 nM to ErbB4-IgG (FIG. 3). Acomparison of HRGβ1₍₁₇₇₋₂₄₄₎ and the thioredoxin HRGβ1₍₁₇₇₋₂₄₄₎ fusionprotein in a competitive binding assay showed that the N-terminalthioredoxin sequence did not affect binding affinity. The ability ofγ-HRG to stimulate tyrosine phosphorylation of ErbB receptor(s) in thehuman breast cancer cell line MCF7, which expresses all tyrosine kinaseclass I receptors in moderate levels (Beerli et al. Mol. Cell. Biol.15:6496-6505 (1995)), was investigated. Cells were treated with variousamounts of γ-HRG and tyrosine kinase activity was determined usingimmunoblotting with anti-phosphotyrosine antibody. Dose dependenttyrosine phosphorylation could be detected. The EC₅₀ was similar to theone seen in MCF7 cells treated with HRGβ1 (60 pM). The functionalactivity of γ-HRG found in the supernatant of MDA-MB-175 cells wasevaluated. The size determination of the secreted isoform was carriedout by Western blot performed with a ErbB2/ErbB4-IgG heterodimer. γ-HRGwas semi-purified from conditioned medium by HPLC using a C4 reversephase column. By SDS-PAGE, a ˜64 kDa band was seen, whereas CHOexpressed full length HRGβ1 migrated with a molecular weight size of 45kDa. These data indicate that the mature protein is 20 kDa greater thanHRGβ1. Without being bound to any one theory, processing of γ-HRG mayhave occurred during the secretion event so as to release the “mature”form of γ-HRG. Binding studies on MCF7 cells revealed a dose dependentdisplacement of ¹²⁵I-HRGβ1₍₁₇₇₋₂₄₄₎ with unpurified conditioned medium.Furthermore tyrosine phosphorylation studies on MCF7 cells also showedsignaling capability of secreted γ-HRG. These findings are in agreementwith the data obtained from experiments carried out with recombinantγ-HRG.

Heregulin is an autocrine growth factor for the human breast tumor cellline MDA-MB-175: MDA-MB-175 cells were treated with an ErbB2 monoclonalantibody (2C4) that interferes with the ligand dependent formation ofErbB2/ErbB3 and ErbB2/ErbB4 heterodimer complexes Sliwkowski et al., J.Biol. Chem. 269:14661-14665 (1994). In a crystal violet staining assay,incubation with 2C4 showed a strong growth inhibitory effect on thiscell line (FIG. 4A). Exogenous HRG did not significantly reverse thisinhibition. On the other hand 2C4 revealed no inhibitory effect on theErbB2 overexpressing cell line SK-BR-3 (FIG. 4B). Treatment with 4D5,another monoclonal antibody against ErbB2 which interacts with adifferent epitope from 2C4 (Fendly et al., supra), was moderate growthinhibitory in MDA-MB-175 cells. Inhibition of cell proliferation by 4D5is dependent on the ErbB2 expression level (Lewis et al. Cancer Immunol.Immunother. 37:255-263 (1993)). A maximum inhibition of 66% in SK-BR-3cells could be detected (FIG. 5B). However this effect could be overcomeby exogenous HRG. To further verify that secreted γ-HRG interacts withErbB receptors in an autocrine manner, additional inhibition experimentswere performed using soluble receptor immunoadhesins. MDA-MB-175 andSK-BR-3 cells were incubated with ErbB4-IgG for 4 days and cellproliferation was measured by crystal violet staining. Treatment withthe immunoadhesin resulted in a strong growth inhibition of 64% inMDA-MB-175 cells. This effect was completely neutralized by addingexcess exogenous HRG. In SK-BR-3 cells, which do not express HRG, theErbB4-IgG treatment was ineffective. From these findings, it wasconcluded that the secretion of γ-HRG by MDA-MB-175 leads to theformation of a constitutive active receptor complex and stimulates thegrowth of these cells in an autocrine manner.

1. A method for activating an ErbB receptor comprising contacting a cellwhich expresses an ErbB receptor with an isolated polypeptide having atleast 95% sequence identity to a polypeptide comprising the aminosequence of SEQ ID NO:4, or SEQ ID NO:10, and wherein the polypeptidestimulates tyrosine phosphorylation of the ErbB receptor.
 2. The methodof claim 1, wherein the polypeptide comprises the amino acid sequence ofSEQ ID NO:4.
 3. The method of claim 1, wherein the polypeptide comprisesthe amino acid sequence of SEQ ID NO:10.
 4. The method of claim 1,wherein the cell is in cell culture.
 5. The method of claim 1, whereinthe cell is mammalian.
 6. The method of claim 5, wherein the mammal ishuman.
 7. A method for enhancing proliferation, differentiation, orsurvival of a cell comprising contacting the cell with a polypeptidehaving at least 95% sequence identity to a polypeptide comprising anamino acid sequence of SEQ ID NO:4, or SEQ ID NO:10, wherein thepolypeptide stimulates tyrosine phosphorylation of a ErbB receptor. 8.The method of claim 7, wherein the cell is a glial cell.
 9. The methodof claim 8, wherein the cell is a human cell.
 10. The method of claim 7,wherein the polypeptide comprises the amino acid sequence of SEQ IDNO:4.
 11. The method of claim 7, wherein the polypeptide comprises theamino acid sequence of SEQ ID NO:10.
 12. The method of claim 7, whereinthe cell is a neuron.
 13. The method of claim 7, wherein the cell is amuscle cell.
 14. A method of treating a neurologic disease or disorderin a subject comprising administering a γ-Heregulin (γ-HRG) polypeptidehaving at least 95% sequence identity to a polypeptide comprising SEQ IDNO:2, SEQ ID NO:4 or SEQ ID NO:10 to the subject, wherein thepolypeptide stimulates tyrosine phosphorylation of an ErbB receptor. 15.The method of claim 14, wherein the neurologic disease or disorder isdue to damage from surgery, trauma, stroke, bulimia, infection,metabolic disease, malignancy, or toxic agents.
 16. The method of claim14, wherein the disease or disorder is amylotrophic lateral sclerosis,Bell's palsy, spinal muscular atrophy, paralysis, Alzheimer's disease,Parkinson's disease, epilepsy, multiple sclerosis, Huntington's chorea,Down's syndrome, nerve deafness, or Meniere's disease.
 17. The method ofclaim 14, wherein the disease or disorder is post-polio syndrome,Charcot-Marie-Tooth disease, Abetalipoproteinemia, Tangier disease,Krabbe's disease, Metachromatic leukodystrophy, Fabry's disease orDejerine-Sottas syndrome.
 18. The method of claim 14, wherein thepolypeptide comprises a sequence of SEQ ID NO:2.
 19. The method of claim14, wherein the polypeptide comprises a sequence of SEQ ID NO:4.
 20. Themethod of claim 14, wherein the polypeptide comprises a sequence of SEQID NO:10.